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Our work shows the efficacy of DNA barcoding for recognizing the early stages of freshwater fish. We collected 3195 larvae and juveniles. Of them, we identified 43 different morphotypes. After DNA barcodes of 350 specimens, we ascertained 7 orders, 12 families, 19 genera, 20 species, and 20 Barcode Index Numbers, corresponding to putative species. For the first time, we reported the presence of the brackish species, Gobiosoma yucatanum in Lake Bacalar. Specimens of the genus Atherinella sp. and Anchoa sp. are possibly new species. Using both methods, morphology, and DNA barcodes, we identified 95% of the total larvae collected (2953 to species, and 78 to genus), and all of them were native. From them, the order Gobiiformes represented 87%. The most abundant species were Lophogobius cyprinoides and Dormitator maculatus, followed by Gobiosoma yucatanum and Ctenobius fasciatus. The Muyil and Chuyanché lagoons have the highest number of species. We present for the first time a short description of Cyprinodon artifrons and Floridichthys polyommus. This information conforms an indispensable baseline for ecological monitoring, to evaluate impacts, and developing management and conservation plans of biodiversity, principally in areas under human pressure such as Sian Ka’an, and Lake Bacalar, where tourism is high and growing in disorder. Inglés