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Assiminea pecos is an endangered species of amphibious gastropod that occupies four widely separated portions of the Rio Grande region in the southwestern United States (Pecos River basin) and northeastern Mexico (Cuatro Cienegas basin). Our statistical and discriminant function analyses of shell variation among the disjunct populations of this species indicate that Mexican specimens differ in their morphometry from those of the United States and can be diagnosed by several characters. We also analyzed variation in the mitochondrial genome by sequencing 658 bp of mitochondrial COI from populations of A. pecos, representatives of the other three North American species of Assiminea, and several outgroups. Our results indicated substantial divergence of the Mexican population of A. pecos, which was consistently depicted as a monophyletic unit nested within or sister to the shallowly structured group comprised of American members of this species. Consistent with our findings, we describe the Mexican population as a new species, which is provisionally placed in the large, worldwide genus Assiminea pending further study of the phylogentic relationships of the North American assimineids. Our molecular data suggest that the Rio Grande region assimineids, which are among the few inland members of the otherwise estuarine subfamily Assimineinae, diverged from coastal progenitors in the late Miocene, with subsequent Pleistocene vicariance of Mexican and American species perhaps associated with development of the modern, lower course of the Rio Grande. [ABSTRACT FROM AUTHOR]