In the present study, we assessed the genetic variation of three Clarias macrocephalus Gu¨nther populations collected from Kedah, Perlis and Kelantan (Peninsular Malaysia) using sequences of partial mitochondrial cytochrome b (Cyt b) and D-loop genes. A total of 57 individuals were sequenced and 1470 bp were obtained (1053 bp Cyt-b; 417 bp D-loop). The analysis revealed 21 haplotypes based on 81 polymorphic sites. Nucleotide diversity (p) was 0.003 in all populations while haplotype diversity ranged from 0.657 to 0.765. No significant genetic differentiation among the three populations was observed. Nevertheless, a number of private haplotypes was discovered, providing valuable information for selective breeding programs.