Accurate taxonomy is essential for conservation, but subspecies-level systematics can be hampered both by a lack of consensus on what constitutes a subspecies and by discordance among data types (e.g., genetics vs. morphology). Here we provide a framework for evaluating subspecies using multidimensional criteria, and suggest that taxa must satisfy multiple criteria to qualify as subspecies. As a case study, we use the Gunnison’s prairie dog (Cynomys gunnisoni), a species for which there has been disagreement regarding the existence of subspecies due to inconsistent application of criteria for defining subspecies. To explicitly test the hypothesis that two subspecies exist, we generated five predictions that could be evaluated with genetic data, while also using morphological and ecological criteria. We sampled 838 Gunnison’s prairie dogs from across the species range and performed a series of genetic analyses using 16 microsatellite and two mitochondrial loci (cytochrome b and the control region). We compared subspecies morphology and quantitatively evaluated whether abiotic and biotic habitat characteristics encountered by each subspecies differed. Genetic results from all five predictions supported the existence of two distinct subspecies within the confines of a proposed revision in the boundary between subspecies. The subspecies differed marginally in morphology and significantly in their habitats, suggesting ecological differentiation. Our results, which are in line with historical descriptions of morphologically distinct subspecies, suggest the subspecies should be recognized. This work provides support for the utility of integrating multiple data and analysis types to inform systematics and conservation.