The genus Chapalichthys (Cyprinodontiformes: Goodeidae) consists of three allopatrically distributed species that occur on the Mesa Central, Mexico. Chapalichthys encaustus primarily occurs in the Río Lerma-Santiago basin, whereas both C. peraticus and C. pardalis have restricted distributions in the adjacent Río Balsas basin. Taxonomic issues in the genus center around the validity of C. peraticus. A formal systematic and taxonomic assessment of the genus inclusive of all three species of Chapalichthys has never been conducted. Therefore, the objectives of this study were two-fold: 1) to assess the phylogenetic relationships among multiple populations and all three species of Chapalichthys using 1,047 bp of mtDNA (ND2) sequence data, and 2) in light of the phylogenetic results, to re-examine the taxonomic status of C. peraticus using meristic and pigmentation characters. The phylogeny indicates two clades, each consisting of a valid species. One clade includes multiple populations of C. encaustus, and a second clade consists of multiple individuals of C. pardalis and C. peraticus. Chapalichthys pardalis and C. peraticus possess nearly identical mitochondrial sequences for ND2. Morphologically, meristic counts of all characters examined showed overlap for all three species and provide no species-specific diagnostic information. Chapalichthys encaustus can be differentiated from C. pardalis and C. peraticus based on the presence of vertical bars along the lateral flank versus a spotted pattern in the other two species. Chapalichthys pardalis and C. peraticus cannot be differentiated from one another based on pigmentation or meristics. The results from this study support the recognition of only two species of Chapalichthys: C. encaustus and C. pardalis.