Diabrotica barberi Smith & Lawrence and Diabrotica longicornis (Say) (Coleoptera: Chrysomelidae) are considered to be sister species, and it has been proposed that the two species may hybridize under field conditions. The objective of this study was to examine genetic and morphological characters of D. barberi and D. longicornis for evidence of field introgression. Both species were collected from sympatric and allopatric areas. Amplified fragment length polymorphisms and morphological characters (color and head capsule width) were used to examine variation within and among populations of D. barberi and D. longicornis. Relatively little of the overall genetic variation was explained by the putative species designation, and most of the genetic variation, both between and within species, was found within populations. In addition, genetic differences were not correlated with geographic location. Beetle color did differ significantly between putative species, with the darkest individuals occurring at the most distal portions of the ranges. Head capsule width varied significantly among populations, but not among species. Both genetic and morphological data support the hypothesis that introgression is occurring between species in the area of sympatry.