It has been proposed that melanism in cordylids evolved in response to a single climatic event and that melanistic populations of Karusasaurus polyzonus are relictual. This study investigates the genetic relationships of melanistic and non-melanistic populations of K. polyzonus along the west coast of South Africa. Thirty-five specimens of K. polyzonus were collected from three ‘melanistic’ and eight ‘non-melanistic’ sample localities. Partial sequence data were derived for two mitochondrial DNA loci (16S rRNA and nicotinamide adenine dinucleotide dehydrogenase component 2 (ND2)) and analysed using maximum parsimony, maximum likelihood and Bayesian inference. In addition, a haplotype network was constructed using TCS and an analysis of molecular variance (AMOVA) was conducted. The derived topologies were highly congruent and showed that melanistic and non-melanistic populations were interdigitated on all tree topologies and a number of haplotypes were shared between melanistic and non-melanistic specimens. These results suggest that melanistic populations of K. polyzonus are ecotypes, not relics.