The Leon Springs pupfish, Cyprinodon bovinus, is federally listed as endangered and currently confined to spring-fed pools on the Diamond Y Spring Preserve in Pecos County, Texas, USA. A refuge population is maintained by U.S. Fish and Wildlife Service in Dexter, New Mexico, USA because of ongoing threats from hybridization with the introduced congener, the sheepshead minnow, C. variegatus. To inform continued conservation and management planning for this species, a comprehensive conservation genomics study using double digest restriction-site associated DNA sequencing was performed. No evidence of contemporary hybridization or recent introgression between C. bovinus and C. variegatus was found. The refuge population was significantly differentiated from wild samples collected at two locations, and the two wild populations (less than 1 km apart) also exhibited significant heterogeneity in allele frequencies. Estimates of within population diversity were smaller for wild locations than the refuge population. Correspondingly, the wild populations’ contemporary effective population size estimates were smaller than the refuge’s effective population size estimates (145–262 versus 679). Temporal analyses, involving genetic data collected from samples obtained in 2013, suggest an increased magnitude of drift acting on the wild populations relative to the reserve population. Based on the results of this study, it appears that steps taken in the early 2000s to eradicate genetically admixed individuals from the wild populations were successful. However, habitat fragmentation and small effective population sizes of the wild populations have led to accelerated drift and decreasing levels of within population genetic diversity. This may jeopardize the long-term persistence of C. bovinus.