The river drainages of Central Mexico have a high degree of freshwater diversity, and are subsequently a focal point for many freshwater fish conservation studies. The livebearing subfamily Goodeinae (Teleostomi: Goodeidae) is a diverse endemic group, under threat from many anthropogenic factors. Xenotoca eiseni, the Redtail Splitfin, a member of this subfamily, has a fragmented distribution in the western basins of the Pacific Coast including the Ríos Grande de Santiago, Compostela, Ayuquila, Coahuayana, and the endorheic Lago de Magdalena and Etzatlán-San Marcos basins. Previous studies have noted high levels of genetic differentiation between the endorheic Lago de Magdalena and Etzatlán-San Marcos basins and surrounding areas which may be indicative of more taxonomic diversity within X. eiseni than currently recognized. The objectives of this study were to use mitochondrial (cytochrome b) and nuclear (ITS-1) DNA sequences and microsatellite data to assess phylogeography, genetic differentiation, and population structure between and within populations of this species. Analysis of the sequence data resulted in two deeply divergent clades, with a mean nucleotide difference of 2.51% within cytochrome b and 0.88% within ITS-1 between populations in the endorheic Lago de Magdalena and Etzatlán-San Marcos basins and all other locations. Microsatellite data also found significant structuring within these two clades of X. eiseni and identified multiple operational conservation units (OCUs). Each of these units contains a proportion of the total variation within the species and requires conservation attention and protection.