The distribution of the rusty crayfish, Orconectes rusticus, has been purported to include large portions of Iowa and Minnesota among many other states, whereas the golden crayfish, O. luteus, has been reported from Kansas, Arkansas, Missouri, and Illinois. Recent collections made in several river basins in Iowa and southern Minnesota and examination of museum specimens collected in Iowa revealed that many records attributed to O. rusticus are in fact O. luteus. We provide a suite of quantitative and qualitative characteristics, both morphological and genetic, that distinguish O. luteus and O. rusticus and also demonstrate that female O. luteus exhibit form alternation. Comparisons of mitochondrial 16S rRNA haplotypes among populations of O. luteus, O. rusticus, O. cf. rusticus, O. placidus, and O. virilis revealed relatively high levels of sequence divergence among taxa as well as within some taxa. We conclude that O. luteus is a native species in Iowa and southern Minnesota with introduced populations of O. rusticus occurring in both states. A more thorough survey of these states, particularly Iowa, is needed to assess the distributions of O. rusticus and O. luteus. If O. rusticus has a relatively restricted distribution in Iowa, efforts to control its anthropogenic and natural dispersal may be more effective if implemented in the near future. Our study indicates the need for more research in taxonomy, even in regions where the fauna is thought to be known well, in order for the conservation of native species and detection and management of nonindigenous species to be successful.