Molecular genetic analyses of populations showing phenotypic variation can provide important insights into the nature and limits of species. This is especially true when interpopulation variation is assessed in a phylogenetic context. The current study applies this approach to evaluate the evolutionary status of the leaf beetle Calligrapha multipunctata variety suturella, a taxon described from a single locality in New Hampshire, but not further discussed in the literature. Here, we report on the collection of material that greatly extends the geographic range of this taxon and on the analysis of cytochrome oxidase I mitochondrial DNA sequences from these specimens and related Calligrapha species. We find that sequences from geographically overlapping populations of C. m. suturella and C. m. bigsbyana form two distinct, phylogenetically divergent clades that exhibit appreciable differentiation (4.5%) at the nucleotide level. This mtDNA differentiation was also evident in each of three localities where both types were collected together on the same host plant. Our results indicate an absence of gene flow between C. m. suturella and C. m. bigsbyana and argue for the recognition of C. m. suturella as a distinct biological species. Accordingly, we elevate its status to C. suturella and provide a detailed diagnosis and description for this new species. Based on an absence of male specimens, a phenotypic similarity to C. m. bigsbyana, and the existence of other parthenogenetic Calligrapha, we speculate that C. suturella may be a parthenogenetic species of hybrid origin.