This study investigated the number of Major Histocompatibility Complex (MHC) class I genes in donkeys, their polymorphism, and their phylogenetic relationship to MHC class I genes of the horse. It has been found through bioinformatic analysis of MHC class I sequences of horses of different haplotypes that classical MHC class I alleles commonly fall within four loci, with additional individual genes that do not fit within any of the four loci. Conserved regions of horse and donkey MHC class I sequences were used to design PCR primers that amplified donkey class I genes. RT-PCR reactions were carried out on donkey lymphocyte cDNA. The MEGA 3 program was used to cluster related sequences and to assign them to individual loci. Donkey sequences followed a similar pattern to that of the horse, having a few loci that contain many of the alleles and additional MHC class I loci that may be unique to one or only a few MHC haplotypes. The donkey alleles fell into either previously established horse loci or loci unique to donkeys. Loci shared between horses and donkeys are probably older loci, having evolved before the split of hoses and donkeys, while loci detected in only one species may be younger. Comparing the allelic variation between horses and donkeys will lead to a better understanding of the evolution of the highly variant MHC region.