It has been assumed that populations of Gambusia geiseri in the Colorado River and Rio Grande basins of Texas were introduced from the San Marcos area of east Texas by humans in the 1930s. This was questionable because of the paucity of early records of Gambusia from the region. Further, a congener, Gambusia speciosa, in the Rio Grande basin carries an apparently “foreign” mitochondrial cytochrome b gene (cytb) haplotype most similar to, but 2% divergent from, a haplotype previously reported for G. geiseri in the San Marcos River. This suggested the populations of G. geiseri outside of the San Marcos area might represent a divergent native lineage of the species. To address this, we assessed variation for cytb and a nuclear sequence (S7 intron 1) in G. geiseri from six spring systems supporting the species. The results suggest nonnative status for all populations of G. geiseri outside the Comal and San Marcos rivers. A phylogenetic analysis of cytb haplotypes in Gambusia species from Texas and northern Mexico demonstrates a sister relationship between G. geiseri and a missing ancestor involved in the mitochondrial ancestry of G. speciosa.