Anopheles funestus Giles (Diptera: Culicidae) is one of Africa’s major malaria vectors. To understand population structure within An. funestus, various molecular markers have recently been developed. We investigated the impact of the Rift Valley on one such molecular marker, a polymerase chain reaction-restriction fragment length polymorphism (RFLP). This system distinguishes An. funestus populations into types M, W, and MW. Generally, these types correlate between geographical location and RFLP type, whereby M associates essentially with eastern Africa, W with western and central Africa, and MW with southern Africa. In total, 606 specimens were screened from 12 countries. Specimens from southern African localities were of the expected MW-type with the exception of the sample from Malawi, which showed all three types. The specimens from Tanzania showed the M- and MW-types, whereas specimens from Kenya showed M- and W-types. We also recorded different RFLP types not recorded previously. Type Y was recorded in Malawi, and Z-type was recorded from four localities: Angola, Malawi, Ghana, and Zambia.