Banija spotted pig breed was created at the end of 19th century by crossing domestic white sows with drooping ears and/or Turopolje pig sows with black Berkshire. After a period of stagnation in breeding, the population is newly recognized only few years ago and is currently in the process of breed valorisation. The aim of this study was to assess genetic diversity and structure of Banija spotted pig (BS) in comparison with geographically close populations, and to assess the differentiation level of Banija spotted pig. Based on the genotypes obtained for 24 ISAG-FAO recommended pig microsatellite markers, basic genetic diversity indicators were determined for 30 samples of BS, 20 samples of Turopolje and Crna Slavonska pig each, and 17 samples of Landrace population. Observed heterozygosity ranged from 0.36 to 0.6 and was high in BS population (0.58). Average allelic richness (range 2 – 5.2) and the rarefacted number of private alleles (range 9 to 27) were the highest in BS. Inbreeding coefficient in BS was estimated 0.06 (bootstrapping confidence interval 0.022 to 0.123). Structure analysis showed that there are five clusters in the four analysed populations, where BS are clearly distinguished from other populations and substructured. Wright’s fixation index estimates are in accordance with the clustering results. In conclusion, the population of Banija spotted pig shows great genetic diversity and is genetically different from neighbouring (geographically close) pig populations.