Gambusia quadruncus n. sp., the llanos mosquitofish, is described from east-central México. The region inhabited by the species represents a hotspot of diversity of Gambusia, and G. quadruncus sometimes coexists with at least three congeners. The species differs from its closest relative, Gambusia affinis, in several characteristics with plausible effects on reproductive isolation, e.g. body size, body and fin morphology, male genital morphology (distal tip of gonopodium) and female anal spot morphology (colouration near the urogenital sinus). Moreover, combined analysis of mitochondrial and nuclear gene sequence data (c. 2158 total base pairs) indicates reciprocal monophyly of G. quadruncus and its sister species G. affinis, with levels of genetic divergence suggesting the two species diverged from one another over a million years ago. The origin of G. quadruncus may reflect a vicariant event associated with Pliocene orogenesis in the Tamaulipas Arch and a frontal section of the Sierra Madre Oriental (Lleran Mesas). Gambusia quadruncus inhabits a variety of freshwater habitats across several river drainages, with its range spanning at least 350 km from north to south, covering over 25 000 km2. A key to aid identification of the species is provided.