Strauzia longipennis (Wiedemann) (Diptera: Tephritidae) is a notoriously variable species. Seven varieties were once recognized. Three varieties were elevated to species status. The status of the other four varieties, including the synonyms for S. longipennis, has been contested. Such taxonomic instability, particularly when associated with variable patterns of host use, suggests that S. longipennis may represent a dynamic complex of host-associated populations in the process of divergence. To detect evidence of genetic differentiation indicating genetically distinct sympatric populations of S. longipennis, we sequenced a fragment of cytochrome c oxidase subunit I of mitochondrial DNA of S. longipennis from two sites (three habitats) in eastern Iowa. At each site, we found two genetically and morphologically distinct sympatric populations. One corresponds to morphological descriptions of S. longipennis variety typica (Loew). The other corresponds to descriptions of S. longipennis variety vittigera (Loew). High levels of genetic differentiation between these divergent sympatric populations suggest the populations might represent host races or incipient species.