In the southwestern United States (US), the Rio Grande chub (Gila pandora) is state-listed as a fish species of greatest conservation need and federally listed as sensitive due to habitat alterations and competition with non-native fishes. Characterizing genetic diversity, genetic population structure, and effective number of breeders will assist with conservation efforts by providing a baseline of genetic metrics. Genetic relatedness within and among G. pandora populations throughout New Mexico was characterized using 11 microsatellite loci among 15 populations in three drainage basins (Rio Grande, Pecos, Canadian). Observed heterozygosity (HO) ranged from 0.71–0.87 and was similar to expected heterozygosity (0.75–0.87). Rio Ojo Caliente (Rio Grande) had the highest allelic richness (AR = 15.09), while Upper Rio Bonito (Pecos) had the lowest allelic richness (AR = 6.75). Genetic differentiation existed among all populations with the lowest genetic variation occurring within the Pecos drainage. STRUCTURE analysis revealed seven genetic clusters. Populations of G. pandora within the upper Rio Grande drainage (Rio Ojo Caliente, Rio Vallecitos, Rio Pueblo de Taos) had high levels of admixture with Q-values ranging from 0.30–0.50. In contrast, populations within the Pecos drainage (Pecos River and Upper Rio Bonito) had low levels of admixture (Q = 0.94 and 0.87, respectively). Estimates of effective number of breeders (N b ) varied from 6.1 (Pecos: Upper Rio Bonito) to 109.7 (Rio Grande: Rio Peñasco) indicating that populations in the Pecos drainage are at risk of extirpation. In the event that management actions are deemed necessary to preserve or increase genetic diversity of G. pandora, consideration must be given as to which populations are selected for translocation.