In the current study, milk protein variation was examined in cattle (Bos indicus), mithun (Bos frontalis), yak (Bos grunniens) and their hybrid populations in Bhutan to estimate genetic variability, conduct genetic characterization and assess the possibility of gene flow between mithun and cattle. Isoelectric focusing of 372 milk samples from 11 populations detected four molecular types of β-lactoglobulin (A, B, E and M), five molecular types of α(S1) -casein (A, B, C, E and X) and three molecular types of k-casein (A, B and X). Mithun and yak shared alleles but were found to exhibit different allele frequencies for the proteins studied. The degree of genetic variability within populations was measured by average heterozygosity and ranged from 24-40% in cattle, 26% for yak and 33% for mithun. We also resolved the traditional mithun and cattle hybridization system via principal component analysis. Our results suggested secondary introgression of mithun genes to the village Thrabum population, and a close genetic relationship between Bhutanese indigenous cattle and Indian cattle.