The western subspecies of the Yellow-billed Cuckoo (Coccyzus americanus occidentalis) has undergone severe population declines during recent years. The current status of this subspecies has been disputed, however, because it cannot be easily separated from C. a. americanus using morphological characteristics. We sequenced most of the cytochrome b gene in five western U.S., three eastern U.S., and two Mexican Yellow-billed Cuckoos, and one Black-billed Cuckoo (C. erythropthalmus) to determine if the subspecies could be diagnosed genotypically. The haplotypes of the eastern and western subspecies differed by four fixed base changes, suggesting that they diverged approximately 205,000–465,000 yr ago. Two of these fixed differences cause amino acid coding changes. Our findings support continued separation of the two subspecies and recognition of the western subspecies as an evolutionarily significant unit.