Domestication centers and the lineages of the wild ass contributing to modern domestic donkey population are still unknown, except the Nubian wild ass. This study is designed to fill the gaps about Anatolian donkey domestication. A total of 68 samples of Anatolian donkeys obtained from Turkey and seven donkeys from Cyprus were analyzed for D-loop and cytochrome b gene regions. Males were also genotyped with three equine Y-chromosomal short tandem repeats (STRs) namely Eca. YM2, Eca.YP9 and Eca.YE1. While the haplotype and nucleotide diversities (h=0.756+/-0.0500 and pi=0.1688+/-0.0012) obtained from D-loop sequences of Anatolian donkey population were found to be moderately high, the Cypriot donkey population were found dramatically reduced (h=0.524+/-0.209 and pi=0.00176 +/- 0.001) than those in Turkey. Only 35% of the Anatolian donkeys were clustered in Clade 1 linked with Nubian ass, while the remaining 65% and 100% of the Cypriot donkeys were clustered in Clade 2. In the present study, Clade 2 was genetically divergent from Somali lineage and from Asiatic wild asses. Thus, they were excluded from to be ancestor of Anatolian and Cypriot donkey populations. A total of 17 and 4 nucleotide differences, discriminating 15 and 6 haplotypes, were defined for D-loop and cytochrome b sequences, respectively. Analyzed Y chromosomal STRs were found to be monomorphic. The allele size of 109, 190 and 195 were identified for Eca. YM2, Eca. YP9 and Eca. YE1, respectively.