Abstract Background Phenotypic and molecular genetic data often provide conflicting patterns of intraspecific relationships confounding phylogenetic inference, particularly among birds where a variety of environmental factors may influence plumage characters. Among diurnal raptors, the taxonomic relationship of Buteo jamaicensis harlani to other B. jamaicensis subspecies has been long debated because of the polytypic nature of the plumage characteristics used in subspecies or species designations. Results To address the evolutionary relationships within this group, we used data from 17 nuclear microsatellite loci, 430 base pairs of the mitochondrial control region, and 829 base pairs of the melanocortin 1 receptor (Mc1r) to investigate molecular genetic differentiation among three B. jamaicensis subspecies (B. j. borealis, B. j. calurus, B. j. harlani). Bayesian clustering analyses of nuclear microsatellite loci showed no significant differences between B. j. harlani and B. j. borealis. Differences observed between B. j. harlani and B. j. borealis in mitochondrial and microsatellite data were equivalent to those found between morphologically similar subspecies, B. j. borealis and B. j. calurus, and estimates of migration rates among all three subspecies were high. No consistent differences were observed in Mc1r data between B. j. harlani and other B. jamaicensis subspecies or between light and dark color morphs within B. j. calurus, suggesting that Mc1r does not play a significant role in B. jamaicensis melanism. Conclusions These data suggest recent interbreeding and gene flow between B. j. harlani and the other B. jamaicensis subspecies examined, providing no support for the historical designation of B. j. harlani as a distinct species.