The present paper aimed to characterize, using 19 microsatellite markers, three native Bulgarian cattle breeds, Iskar, Rhodope Shorthorn and Bulgarian Rhodope and to clarify their population structure. The three breeds own a genetic variability comparable with other European cattle breeds, nevertheless they showed a significant deviation from Hardy-Weinberg equilibrium in terms of heterozygote deficiency. Allelic frequencies distribution among breeds was highly significant confirming their genetic uniqueness. The population structure of Rhodope Shorthorn was complex and is probably the cause of its rather high FIS estimate (0.111); Iskar breed structure is also rather fragmented and should be studied more deeply while Bulgarian Rhodope population seemed to be the less variable. Presented results helped to clarify the present situation of Bulgarian cattle biodiversity giving interesting suggestions for their management and conservation.