The Cuatro Ciénegas killifish Lucania interioris is an endemic fundulid in the Chihuahuan Desert valley of Cuatro Ciénegas, Coahuila, México. It occurs in 3 major drainage regions of the basin and is limited primarily to naturally fragmented and physicochemically severe habitats of the valley floor. We assessed mitochondrial DNA variation and population genetic structure in L. interioris from 8 populations representative of the entire geographic range of this species, including its distribution within and among major drainage regions (western, central, and southeastern) of the valley. Five populations were invariant, including both populations in the western region (Haplotype A only), whereas 2 haplotypes were observed in each of the remaining populations. Haplotype diversity was low in the central basin (H_D = 0.10 and H_D = 0.19) and moderate in the southeastern basin (H_D = 0.52). Low levels of divergence among haplotypes (0.3%–0.9%), together with high levels of genetic differentiation among regions (F_CT = 0.847, P < 0.001), indicate complete isolation among major drainage regions. These results agree with previous documentation of morphological differentiation between populations from the western and central basins. Populations in the southeastern basin are divergent genetically and, therefore, also may exhibit morphological distinction.