The Davis Mountains cottontail, Sylvilagus robustus, is morphologically different from the eastern cottontail, S. floridanus, but previous genetic analysis of mitochondrial DNA data did not recover 2 genetically distinct groups. Our study used a nuclear DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), to test the hypothesis that S. robustus is genetically distinct from S. floridanus. We tentatively considered any individual collected at an elevation >1,400 m as S. robustus and later confirmed our identifications with morphological or genetic data, or both. Principal component and discriminant function analyses of 6 previously published cranial measurements confirmed morphological distinctiveness. For genetic analyses we analyzed 273 AFLP fragments from 20 individuals of S. robustus and compared them to 16 S. floridanus, 4 S. audubonii, and 1 S. obscurus. Results from phylogenetic and population genetic analyses suggest a significant lack of gene flow between the 2 species. Together, these data support recognition of S. robustus as a separate species.