The host plant range of pests can have important consequences for its evolution, and plays a critical role in the emergence and spread of a new pest outbreak. This study addresses the ecological genetics of the indigenous African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae), in an attempt to investigate the evolutionary forces that may be involved in the recent host range expansion and establishment of this species in Ethiopian and southern African sugarcane. We used populations from Ethiopia, Zimbabwe, and South Africa to examine whether the host range expansion patterns shared by the Ethiopian and the southern African populations of B. fusca have evolved independently. Base-pair differences in the cytochrome oxidase I (COI) gene were used to characterize haplotype diversity and phylogenetic relationships. There were seven haplotypes among the 30 sequenced individuals collected on four host plant species from 17 localities in the four countries. Of the seven COI haplotypes identified, the two major ones occurred in both sugarcane and maize. Genetic analyses revealed no detectable genetic differentiation between southern African B. fusca populations from maize and sugarcane (FST = 0.019; P = 0.24). However, there was strong evidence of variation in genetic composition between populations of the pest from different geographic regions (FST = 0.948; P < 0.001). The main implication of these findings is that the B. fusca populations in maize in southern Africa are more likely to shift to sugarcane, suggesting that ecological opportunity is an important factor in host plant range expansion by a pest.