Equine mitochondrial DNA (mtDNA) phylogeny reconstruction reveals a complex pattern of variation unlike that seen in other large domesticates. It is likely that this pattern reflects a process of multiple and repeated, although not necessarily independent, domestication events. Until now, no clear geographic affiliation of clades has been apparent. In this study, amova analyses have revealed a significant non-random distribution of the diversity among equine populations when seven newly sequenced Eurasian populations were examined in the context of previously published sequences. The association of Eastern mtDNA types in haplogroup F was highly significant using Fisher's exact test of independence (P = 0.00000). For the first time, clear biogeographic partitioning has been detected in equine mtDNA sequence.