The pyralid moth Eldana saccharina Walker is an indigenous insect widely distributed throughout sub-Saharan Africa. Studies have shown that populations from West Africa have distinct behavioural differences compared to populations from East and southern Africa. In addition, the parasitoid guilds attacking populations in these different regions are markedly different. This marked geographical variation evoked a hypothesis of genetic differentiation. To evaluate this hypothesis a molecular analysis was conducted on populations of E. saccharina from throughout much of the species' range, using the cytochrome coxidase subunit I (COI) region of the mitochondrial genome. A minimum spanning network and a maximum parsimony tree separated the 21 specimens into three distinct groups. Results revealed the presence of substantial genetic differentiation that is related to geographic variation.