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The aim of the study is to increase the knowledge on the genetic structure status of the autochthonous Slavonian Syrmian Podolian cattle breed currently listed by the FAO as “critically endangered”. Blood samples obtained from a total of 20 randomly selected individua were included in genotyping upon microsatellite panel recommended by ISAG and FAO. The procedure enabled the identification of as many as 214 (96.34%) genotypes and, at the same time, 129 allelic variations were found with X= 11.73 alleles per locus. The genotypes showed differences between the microsatellite loci and also significant differences (p< 0.05) in the frequency of both homozygosity and heterozygosity. The analyses of the allelic distribution within microsatellite loci showed marked variability. The distribution of genetic relationship was expressed by Wright’s F coefficients. A moderate level of genetic diversity was observed at the tested loci. The findings demonstrate the degree of breed relationship and breed purity but also indicate that gene pool of the tested population has been threatened. Therefore, urgent measures must be taken to support both the genetic consolidation and preservation of the remaining population of Slavonian Syrmian Podolian cattle in Croatia.