The fathead minnow (Pimephales promelas) is a key model of vertebrate toxicity. Standardized tests of toxicity in fathead minnow have been developed to support regulatory science, and much is known about the response of the species to various environmental pollutants. However, there is little data on genetic variation within the species, despite the potential influence of genetic background on toxicological outcomes. Furthermore, the phylogenetic relationships among Pimephales species are not fully established and rates of evolutionary divergence within the species and genus have not been investigated. This study examined patterns of genetic variation across the genome within a single wild population of fathead minnow from the San Juan River of the southwestern U.S. These patterns were contrasted with variation in an inbred, captive population reared by the U.S. Environmental Protection Agency. The study also examined variation in two specimens each of the congeners bluntnose minnow (P. notatus), slim minnow (P. tenellus), and the bullhead minnow (P. vigilax). This data release describes the availability of the raw sequence data, the methods for identifying genetic polymorphisms among the samples, and provides the called polymorphisms in a standardized format (the variant call format, or VCF).