Three wild populations of Clarias gariepinus from Esaodo (River Osun), Owena (River Owena), and Agbabu (River Oluwa), and three farmed populations, viz: Akure, Ilesa and Ado-Ekiti, in Southwest Nigeria, were analysed for their genetic differences using Random amplified polymorphic DNA (RAPD) analysis. Live specimens comprising 40 individuals (680 ± 3.28 g) from each location were collected and kept in six concrete tanks (2x1x1m). Altogether 435 reproducible bands were obtained from six populations for the nine RAPD primers used. The Analysis of Molecular Variance (AMOVA) indicated that the sampled populations are significantly different from each other, and that 99% of the total variation resided within the population. The percentage of genetic identity (GI) of RAPD-PCR profile among six populations ranged from 74.6% to 83.5%, while Genetic distance of the six populations based on RAPD-PCR profile ranged from 0.180 to 0.293. Estimates of genetic variation in wild and cultured populations of C. gariepinus were made, and total and mean number of segregating fragments were 71 (89.9%), 34.5 and 59 (74.7%), 35.4, respectively. Total gene diversity within wild and cultured populations (Ht) was 0.3419 and 0.3010, respectively. The study established that there is genetic variability in both wild and cultured C. gariepinus. RAPD showed that samples within the wild and cultured populations under study were closer to each other than between the two habitats. With reference to total gene diversity values and total number of segregating fragments, the wild population was considerably more diverse than the cultured population.