Pangasius macronema is an endemic and important species with high economic value for fisheries in the Mekong River basin. The rapid increase in fishing levels can have an impact on genetic diversity. This study evaluated the genetic diversity of three wild populations of P. macronema using intersimple sequence repeat (ISSR) markers. Six primers were used to analyze 72 samples from three locations (populations) along the Hau and Tien Rivers in An Giang (n= 20), Soc Trang (n= 29), and Dong Thap (n= 23). The results showed that all populations of P. macronema had relatively high genetic diversity (effective number of alleles, Ne, from 1.381 to 1.454 and unbiased heterozygosity, uHe, from 0.243 to 0.286), with the Soc Trang population having the highest level. As a result, Nei's unbiased genetic distances among the three populations were low, ranging from 0.015 to 0.035, with the smallest genetic distance between the An Giang and Dong Thap populations. The genetic differences among the three populations may be influenced by the species’ migration patterns and hydrological connectivity.