Many species are currently undergoing reductions in population size due to widespread habitat loss and expanding human activities. Because interspecific hybridization is often a consequence of population decline and fragmentation, identification of individuals or populations with hybrid ancestry is an increasingly important issue in conservation biology. In many wild cattle and bison species, the problem of natural hybridization has been compounded by indiscriminate crossbreeding with domestic cattle for the purpose of improving domesticated stocks. Therefore, a genetic test using the polymerase chain reaction was developed so that wild cattle and bison with domestic cattle mitochondrial DNA (mtDNA) haplotypes could be rapidly identified. Using this genetic test, domestic cattle mtDNA haplotypes were detected in Bos grunniens (yak), Bison bonasus (European bison), and 6 out of the 15 (40%) Bison bison (North American bison) populations tested. In total, 30 out of the 572 (5.2%) North American bison tested, were found to have domestic cattle mtDNA. The hybrid origin of these mtDNA haplotypes was verified in a phylogenetic analysis using the parametric bootstrap. These results are discussed in terms of their implications for the conservation status and future management of wild cattle and bison species.