In 2018, the Banija spotted pig breed (Banijska šara, BS) was added to the list of native and protected pig breeds and strains. This, along with the fact that the population is small in comparison, confirms that information about its phenotypic and genetic traits is required for the implementation of targeted pairings and conservation programs aimed at preserving genetic diversity and breed characteristics within the population. Identifying potential genes for litter size increases the efficiency of reproduction, making litter size one of the most significant production characteristics in pig production. The extended goal of this research was to determine morphometric characteristics, external features, reproductive indicators, and the polymorphism of the ESR1 and RBP4 genes, along with their impact on litter size, genetic diversity, and population structure of BS pigs. Body mass and morphometric properties (wither’s height, height at the sacrum, and root of the tail; seven measurements on the trunk and four on the extremities) were determined on fifty adult and reproductively active individuals (38 sows and 12 boars), aged 14 to 44 months, in various reproductive stages. For morphometric measurements, Lydtin’s rod, a measuring tape, and a protractor were utilised. A livestock scale (TehnoNet) was used to determine body mass. In order to compare the reproductive indicators of the Banija spotted pig with those of two other autochthonous breeds (Black Slavonian and Turopolje pigs), an analysis was conducted on 184 litters of Banija spotted pigs, 1174 litters of Turoplje pigs, and 18228 litters of Black Slavonian pigs. DNA isolation was done on twenty individuals of the Turopolje, Black Slavonian, and Landrace pig breeds, as well as on reproductively active, non-related Banija spotted sows and boars that were included in the study (N=30). 24 MS and 16 MS markers were selected from the ISAG-FAO recommendation list. 25 Banija spotted pigs were used for the genotyping of the candidate genes for litter size (ESR1 and RBP4), with at least one litter recorded and assessed using the PCR-RFLP method. The average body mass of the sows and boars that were measured was roughly the same (162.6 kg vs. 162.2 kg), with the sows having slightly bigger variances between the minimum and maximum values (209 kg) than the boars (155 kg). Sows measured 72.1 cm at the withers, which was 5 cm less than boars’ average height. Boars had values that were, on average, 5-7 cm higher than sows for both the height at the withers and the height to the base of the tail. The sows’ average carcass length was 116.3 cm, which was roughly 2 cm less than the boars’. Chest circumference (131.1 cm or +7 cm), abdominal circumference (132.6 cm or +7.4 cm), and chest width (33.6 cm or +0.3 cm) were all greater in sows than in boars. The average head length and head width measured in sows were 31.1 cm and 16.1 cm, respectively. The average nose length of sows was found to be shorter (-0.7 cm) than that of boars, whereas their average nose width was slightly greater (+0.3 cm). Comparing male categories (boars and young boars) to female categories (sows and gilts), the measured values of tail length were greater in the former. With respect to ear position, the majority of the individuals under analysis had flat ears, with semi-flat ears following closely behind. Only one male (a boar) showed projecting ears. In terms of back line appearance, 91.7% of sows and all other categories (boars, gilts, and young boars) have a straight back line. Only 8.3% of sows had a convex dorsal line. Regarding the nasal profile of BS pigs, 56.8% of sows and 66.7% of boars were found to have a flat nasal profile. On the other hand, sows accounted for 43.2% and boars for 33.0% of the total number of individuals with a sunken nasal profile. Of the sows, 55.6% had short hair and 25% had boars; of the sows, 44.4% had long hair and 75% of the boars had short hair. Of the sows, 52.8% had black pigment on their hooves, while the boars had 8.3%. 52.8% of sows and 58.3% of boars had a flat tail, according to an analysis of the tail’s appearance in several BS pig groups. On average, 51.9% of the body surface was covered in black patterns, compared to 48.1% in white patterns. In 50% of boars with the right ear and 66.7% of boars with the left, the pigment content was undetermined. For both the left and right ears, more than one-third of the coloured ear was present in 64.9% and 62.2% of the sows, respectively. The average litter size of 184 BS pigs, or 8.5 total born (TB) or 7.5 born alive (FL) piglets, was found by analysis of the observed litters. A slightly greater proportion of FL female piglets (3.9) than male piglets (3.6) was discovered after the sexes’ relationship was analysed. There were statistically significantly lower values (P<0.05) of all observed fertility indicators in the two other original breeds of pigs in the Republic of Croatia, the Turopolje pig (TP) and the Black Slavonian pig (BSP), as compared to BS. Genotype frequencies for both examined marker genes (ESR1 and RBP4) in the BS pig population do not show significant deviation from the Hardy-Weiberg equilibrium. The ESR1 gene is characterised by a high proportion of allele A (0.78), i.e., a high proportion of homozygous individuals of the AA genotype (0.60) and heterozygous AB (0.36), while only one individual had the BB genotype (0.04). Allele B (0.60) is the most common and accounts for the biggest proportion of genotypes with BB (0.44), according to the study of RBP4 gene polymorphism. Next in frequency (0.32) are heterozygotes, with the AA of homozygous individuals having the lowest number observed. In BS pigs, the polymorphism of the ESR1 gene did not significantly affect fertility indicators; however, in primiparous sows, this influence is evident with respect to the RBP4 gene. For the purposes of defining the genetic diversity of the BS pig breed and its differentiation in relation to other native and pure pig breeds in Croatia, an analysis of genetic variability and comparison with Turopolje pigs (TP), and Black Slavonian pigs (BSP), and pure pig breeds bred in the Republic of Croatia, Landrace (L), Large Yorkshire (LY), Duroc (D), and Pietrain (P) were done. According to the findings, BS had the highest average number of alleles per locus (7.36), whereas TP had the lowest average (2.92). In Y (0.7221), the observed heterozygosity (Ho) was highest, whereas in TP (0.342), it was lowest. The inbreeding coefficient was the highest for TP (0.08830), slightly lower for BS (0.08691) and the lowest for D (-0.01172). The highest FST value for the Banija spotted pig breed was 0.2868 (between BS and TP), while the lowest was 0.1052 between BS and BSP. Of the pure breeds, the Pietrain (0.2492) is the farthest from it, and the Landrace (0.206) is the closest. An average of 578 animals was genotyped using 16 MS markers, 127 of which were BS. The observed heterozygosity (Ho) in the BS was 0.6349, and the expected heterozygosity (He) was 0.6566, while the lowest values were recorded in the Turopolje pigs (Ho 0.369; He 0.3787), and the highest in the Large Yorkshire pigs (Ho 0, 7221; He 0.7059). The lowest FST value was recorded between BS and L (0.0878), while the highest was 0.3112 (between BS and TP). Out of all the native pig breeds, BS and BSP had the closest fixation index value, at 0.1079. As a result, the BS pig population exhibits high genetic diversity within itself and differs genetically from other similar and geographically close pig populations, such as the Black Slavonian or Turopolje pigs, with whom it shares common ancestors.