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The allozyme variation and population genetic structure of Horabagrus brachysoma in three natural populations from the southern part of the Western Ghats region, India, were investigated by polyacrylamide gel electrophoresis. Variations at 14 loci from 14 enzyme systems were analyzed. The allozyme analysis revealed a high level of genetic variation in this species, with an average of observed alleles per locus of 2.357 and observed heterozygosity of 0.178. The positive value of the fixation index (FIS = 0.507) implied a significant deficiency of heterozygosity at the population level. The highly significant probability (P < 0.0001) for the overall loci suggested that the three sample sets were not part of the same gene pool.