Abstract: To investigate genetic variation four hatchery populations (Shambhuganj, Brahmaputra, Anudan and Bhai-Bhai) of Thai pangas (Pangasius hypophthalmus) of Mymensingh region in Bangladesh were analyzed by allozyme electrophoresis. Using six enzymes, nine presumptive loci were identified where three (Est-1*, Gpi-2* and Pgm*) were polymorphic (p,0.95) in Anudan and Brahmaputra populations and one locus, Est-1* was polymorphic in Shambhuganj population. No polymorphic locus was found in Bhai-Bhai population. The mean proportion of heterozygous loci per individual was 11.3±8.5% which ranged from 0.0% (Bhai-Bhai population) to 20% (Anudan population). The mean number of allele per locus was 1.3 that ranged from 1.0 (Bhai-Bhai population) to 1.6 (Anudan population). The UPGMA dendrogram showed that the four populations were divided into two groups by the genetic distance, D=0.18 but the populations were not geographically isolated. Finally the results indicated that the Anudan and Brahmaputra populations were showed higher genetic variation whereas Bhai-Bhai population lost their genetic variation completely.