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The detection of hybridization among freshwater fish species is of main concern for conservation programs and fish farming. We assessed the incidence of natural hybridization between two vulnerable species of Andean pupfishes (Cyprinodontidae; Orestias agassizii and O. luteus) that represent an important component of local fisheries. We combined mitochondrial and nuclear DNA sequencing, microsatellites genotyping and morphometrics to characterize hybridization patterns between the two species in lakes Titicaca and Uru Uru (N = 175). The clustering analysis of 10 microsatellite loci together with heterozygosity distribution at seven species-specific diagnostic sites in rhodopsin (nuclear DNA) proved to be a robust diagnostic tool to detect F1 and potential backcross hybrids. For the first time on a genetic basis, we confirmed the incidence of natural hybridization between the two Andean pupfishes, at frequency rates reaching almost 10 %. The morphological intermediacy criterion (relative to parental species) did not apply in hybrids, since the latter (i) deviated through their larger caudal peduncle height, and (ii) had greater, overlapping ranges in their morphometric ratios. Although viable F1 and F2 hybrids between O. agassizii and O. luteus had been reported under controlled conditions, the ecological and/or demographic factors facilitating interspecific gene flow in the wild remain speculative. We recommend that regional fish farming and restocking programs targeting O. agassizii and O. luteus call on the genetic diagnosis of hybrids to avoid the potentially deleterious impacts of releasing hybrid populations in the wild.