Gambusia nobilis is a federally Endangered species found across a fragmented distribution within the Pecos River Drainage of Texas and New Mexico, USA. Drought, human water usage, and potential hybridization and competition with introduced congeners threaten species persistence. Therefore, a population genomics study was conducted to provide critical information for conservation planning. Unsupervised clustering suggested hierarchical structure, with a primary K =3, and deep divergences were detected among samples grouped into the Leon Creek watershed, the Toyah Creek watershed, and water bodies within the Bitter Lake National Wildlife Refuge (F’ST = 0.55–0.76 for putatively neutral data). Phylogenetic analyses showed three distinct clades corresponding to these groups, with split times estimated to be in the last 50,000 years. Subsequent morphological analyses detected differences among the three groups, including male colour pattern in life, and the number of caudal-fin rays in both sexes. Taken as a whole, the results indicate that the endangered G nobilis comprises three species (two of which are named herein), rather than one, and the study highlights the daunting yet critical task of describing species diversity during a period of unprecedented diversity loss.