The Pánuco-Tamesí complex in eastern Mexico is globally recognized as an important ecoregion due to its high level of endemism. In this study, DNA barcodes were generated for 152 individuals of 31 species. Additionally, 170 DNA barcodes for the related species available in the Barcode of Life Database (BOLD) system were included to test the ability of barcoding technique to discriminate between the closely related species. DNA barcoding allowed the discrimination of 79.2% of the analyzed species; poor resolution was observed in four genera in which the levels of resolution ranged from 16.6% in the genus Herichthys to 77.7% in the genus Xiphophorus. The results of this study demonstrate that DNA barcoding is a useful exploratory tool but fails to discriminate between closely related species.