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Oreochromis niloticus is the most important aquacultural species worldwide. Several countries are interested in repopulating natural or artificial reservoirs with the fry of tilapia (Oreochromis sp.) to develop new fisheries or to enhance the existing ones. However, as tilapia is considered an invasive species, it is essential to check the existence of the species in the place before releasing any fry. Characterization of tilapia species with meristic is unreliable because of existing morphological similarities among species. In this work, the composition of the tilapia species of Laguna El Mastranzo (LEM), Nayarit, Mexico, was explored for the first time through a molecular method using the mitochondrial DNA control region. Three tilapia species (Oreochromis niloticus, O. aureus, and O. urolepis) were found in LEM, and it was confirmed that the two commercial strains (Aquamol and Spring) intended for repopulation were O. niloticus. Seven haplotypes of mitochondrial DNA control region sequences were obtained: three in LEM, three in Aquamol, and one in Spring strains. Before repopulating any reservoir, a molecular verification strategy is recommended to avoid spreading the species. The results of this work represent a glimpse into the genetic material of LEM, Nayarit, Mexico.