Bighead catfish (Clarias macrocephalus) is an important freshwater species for aquaculture and fisheries in Southeast Asia. The species has been domesticated for more than 30 generations in hatcheries, but wild population numbers have declined to “threatened” levels. In this study, genetic diversity was quantified in areas of intensive catfish culture and natural areas of the Mekong Delta, Viet Nam to inform hatchery genetic improvement programs and conservation actions. Fish were collected from three hatcheries and four wild populations in conservation parks (wild-conservation) and seven wild populations in rice fields of aquaculture-intensive areas (wild-farming). Results based on six microsatellite loci showed all populations had relatively high levels of genetic diversity for allelic richness (range 8.10– 10.18), and observed heterozygosity (range 0.708– 0.819). However, genetic diversity was lower in hatchery fish and wild farming populations as evidenced by significantly lower (P ≤ 0.03) allelic richness (9.16 ± 0.27 and 9.27 ± 0.66, respectively) than wild conserved populations (10.01 ± 0.19). Pairwise individuals’ genetic relatedness within three cultured populations (0.013–0.028) and five wild farming populations (0.016–0.039) indicated low potential for inbreeding in the immediate future. Genetic differentiation among populations (FST = 0.017 ± 0.002) indicated a modest level of gene flow, likely due to the expansive river network, straying of hatchery fish, and large-scale transport of wild fish to hatcheries.