Genetic variation in three Croatian donkey populations, Istrian (IS), North Adriatic (NA) and LittoralβDinaric (LD), was analysed using eight microsatellite loci and by sequence and SSCP analysis of the proximal portion of the mtDNA Dβloop region. The analysis of microsatellite loci revealed observed heterozygosities in the range of 0.37 (MPZ002 in LD) to 0.85 (AHT21 in LD) and polymorphic information content values in the range of 0.36 (MPZ002 in NA) to 0.78 (AHT21 in LD). The overall probability of exclusion was 0.991. Two populations (IS and NA) were closely related (Fst=0.0034), whereas genetic distances between IS and LD (Fst=0.021) and NA and LD (Fst=0.027) were higher. Using AMOVA, 97.6% of the total genetic variance was portioned within populations, while 2.7% was portioned between the LittoralβDinaric population and the Istrian/North Adriatic population group. Sequencing of the proximal part of the mtDNA Dβloop region revealed 36 polymorphic sites representing 19 haplotypes which clustered into three haplotype groups (Y, W, Ws). Only the Y haplotype was found in the IS population which is characterized by a large body size. Haplotypes W and Ws were found in the NA and LD populations which include smaller animals. All three haplotypes were found in the LD population, indicating sporadic migration events from the IS into LD donkey population.