The patterns of genetic variability and genetic differentiation among five wild and ten hatchery populations of Catla catla – an economically important South Asian freshwater fish were assessed using 15 microsatellite markers in a total 750 individuals (n = 50 individuals per population) in this study. The level of genetic diversity in terms of the average allelic richness (Ar), alleles number (Na), number of effective alleles (Nae) and heterozygosity (H) was observed low-to-moderate in the hatchery while moderate in the natural populations of C. catla. The highest mean values of Na, Ne, Ar and H were found in the wild populations in comparison to the hatchery populations in the present study. The mean values of Na, Ar, Ne and Ho ranged from 2.80 to 4.00, 2.80 to 3.988, 2.412 to 2.859 and 0.462 to 0.524, respectively were noted in hatchery populations of C. catla. The same varied from 3.867 to 4.467, 3.867 to 4.465, 2.694 to 3.521 and 0.553 to 0.693, respectively in the natural populations of C. catla.The average of expected heterozygosity (He) values were noted higher as compared to the Ho. On the average, the values of inbreeding coefficient (FIS) in hatchery populations were found positive, but one wild population showed negative mean value too. On average, the FIS values ranged from 0.0872 to 0.2042 and from -0.045 to 0.164 in the examined hatchery and natural populations of C. catla, respectively. After correction of significance levels for 150 simultaneous tests (P < 0.05), only one instance in domesticated stocks while thirteen tests out of 75 tests in natural populations of C. catla were found to depart from Hardy-Weinberg Equilibrium (HWE) in the present study. The pairwise estimates of FST revealed limited genetic differentiation between hatchery but low-to-moderate between wild populations. Among pairs of hatchery and natural populations of C. catla, the unbiased genetic distance indicated considerable variation (P<0.05) in magnitude. The AMOVA revealed that most of the variation was found within individuals in both hatchery and wild populations. The AMOVA specified that 4.869% variation was contributed due to variation between hatchery populations while 6.3054% due to variation between natural populations of C. catla. Analysis of genetic relatedness among all the examined hatchery and natural populations was estimated by constructing UPGMA dendrogram and STRUCTURE admixture model which predicted that the populations in the same clusters had a close genetic relationship. The present study inferences would be helpful in resolving the genetic issues relating to C. catla re-stocking plans and brood stock management practices in future.