The purposes of this research were to identify fish species using DNA barcodes or partial sequences of cytochrome b (Cytb) and to assess the diversity of fish in the Mae Tam reservoir, Phayao province, Thailand. Fish samples were collected 3 times, during the winter, summer, andrainy seasons, from 2 sampling sites using gillnets with 3 mesh sizes (30, 50, and 70 mm). A total of 34 representative samples were classified into 12 species, 7 families and 6 orders bymorphological-and DNA barcoding-based identifications. However, one cichlid species, Cichlasoma trimaculatum, could only be identified using DNA barcoding. Family Cyprinidae had the greatest diversity, 50.00%. The diversity, richness and evenness indices ranged from 0.43-0.65, 0.64-1.46, and 0.27-0.40, respectively, indicating that fish diversity at both sampling sites was relatively low. A comparison of the catch per unit effort (CPUE) with 3 different mesh sizes found that the 50 mm mesh size was the best (474.80±171.56 g/100 m 2/night), followed by the 70 mm (417.41±176.24 g/100 m 2/night) and 30 mm mesh sizes (327.88±115.60 g/100 m 2/night). These results indicate that DNA barcoding is a powerful tool for species identification. Our data can be used for planning the sustainable management of fisheries resources in the Mae Tam reservoir.