Hybrid speciation may contribute significantly to generating biodiversity, but only a few well-documented examples for it exist so far that do not involve polyploidization as a mechanism. The swordtail fish, Xiphophorus clemenciae, shows common hall-marks of a hybrid origin and still overlaps in its current geographic distribution with its putative ancestral species (X. helleri and X. maculatus). Xiphophorus clemenciae provides an ideal system for investigating the possible continued genetic interactions between a hybrid and its parental species. Here we use microsatellite and mitochondrial markers to investigate the population structure of these species of swordtails and search for signs of recent hybridization. Individuals were sampled from 21 localities across the known range of X. clemenciae – the Isthmus of Tehuantepec (IT) Mexico, and several environmental parameters that might represent barriers to dispersal were recorded. The hybridization event that gave rise to X. clemenciae appears to be rather ancient and a single origin is likely. We find negligible evidence for ongoing hybridization and introgression between the putative ancestral species, since they now occupy distinct ecological niches, and a common haplotype is shared by most populations of X. clemenciae. The population structure within these species shows an isolation-by-distance (IBD) pattern and genetic differentiation between most populations is significant and high. We infer that tectonic evolution in the Isthmus has greatly restricted gene flow between the southern and central IT populations of X. clemenciae and X. helleri and provide preliminary information to aid in conservation management of this geographically restricted hybrid species, X. clemenciae.