The breeding distribution of Painted Buntings (Passerina ciris) is comprised of two allopatric populations separated by a 550-km distributional gap in the southeastern United States. Curiously, the boundary between the two recognized P. ciris subspecies does not separate the two allopatric breeding populations but instead runs roughly through the center of the interior population. Genetic relationships among these subspecies, and the allopatric breeding populations of Painted Bunting, have not been assessed. Given the recent decline in overall abundance of this species, such an assessment is warranted. We sampled birds from 15 localities (138 individuals) and identified 35 distinct haplotypes, six belonging to the Atlantic Coast population and 26 to the interior population, with three shared by both populations. AMOVA results showed that a significantly greater portion of the total genetic variance is explained when grouping birds by the interior and Atlantic Coast populations rather than by subspecies. Furthermore, our data indicate that the Atlantic Coast and interior populations represent independently evolving taxa, with no measureable gene flow between them. Although recently diverged (26,000-115,000 years ago), these isolated bunting populations represent incipient species. For development of conservation strategies, we suggest that the Atlantic Coast and interior populations be recognized as separate management units. ?? 2011 Springer Science+Business Media B.V.