The African sugarcane stalk borer, Eldona saccharina Walker (Lepidoptera: Pyralidae), is widely distributed throughout sub-Saharan Africa and is an important insect pest of maize and sugarcane. The insect shows significant variation in behaviour, host plant and natural enemy guild in different regions. Several attempts to redistribute the natural enemies of E. saccharina from West Africa to South Africa were unsuccessful. The significant behavioural, host plant and natural enemy variations as well as failures of biocontrol attempts evoked a hypothesis of genetic diversification. To evaluate this hypothesis a molecular analysis was conducted on geographically isolated populations of E. saccharina from East, North, South and West Africa, using the cytochrome c oxidase subunit I (COI) region of the mitochondrial genome. The results revealed that E. saccharina populations are separated into four major units corresponding to the West Africa, Rift Valley, South/East Africa and southern African populations. Mitochondrial DNA divergence among the four populations ranged from 1% to 4.98%. To examine the impact of the observed genetic variation on the fertility of inter-population crosses, a mating experiment was conducted between the Rift valley and South African population to produce an F1 generation, and these were backcrossed with the South African parent population. Fertility of eggs produced by the F1/parent population cross was significantly reduced when compared to fertility of the "true" South African line, and the F1/F1 cross. The contributions of the observed genetic differences and inter population incompatibility for the failure of previous biocontrol attempts are discussed and recommendations on future biocontrol practices are given.