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Background   Anopheles funestus s.s. , one of the major malaria vectors in sub-Saharan Africa, belongs to a group of eleven African species that are morphologically similar at the adult stage, most of which do not transmit malaria. The population structure of  An. funestus  based on mitochondrial DNA data led to the description of two cryptic subdivisions, clade I widespread throughout Africa and clade II known only from Mozambique and Madagascar. In this study, we investigated five common members of the  Anopheles funestus  group in southern Africa in order to determine relationships within and between species.    Methods  A total of 155 specimens of  An. funestus ,  An. parensis ,  An. vaneedeni ,  An. funestus -like and  An. rivulorum  from South Africa, Mozambique and Malawi were used for the study. The population genetic structure was assessed within and between populations using mitochondrial DNA.    Results  The phylogenetic trees revealed three main lineages: 1)  An. rivulorum ; 2)  An. funestus -like clade I and  An. parensis  clade II; and 3)  An. funestus  clades I and II,  An. funestus -like clade II,  An. parensis  clade I and  An. vaneedeni  clades I and II. Within  An. funestus , 32 specimens from Mozambique consisted of 40.6% clade I and 59.4% clade II while all 21 individuals from Malawi were clade I. In the analysis of mitochondrial DNA sequences, there were 37 polymorphic sites and 9 fixed different nucleotides for  ND5  and 21 polymorphic sites and 6 fixed different nucleotides for  COI  between the two  An. funestus  clades. The results for  COI  supported the  ND5  analysis.    Conclusion  This is the first report comparing  An. funestus  group species including  An. funestus  clades I and II and the new species  An. funestus -like.  Anopheles funestus  clade I is separated from the rest of the members of the  An. funestus  subgroup and  An. funestus -like is distinctly distributed from the other species in this study. However, there were two clades for  An. funestus -like,  An. parensis  and  An. vaneedeni . Further investigations are needed to determine what these results mean in terms of the specific status of the clades within each taxon and whether this has any epidemiological implications for malaria transmission.

Population genetic structure of the major malaria vector Anopheles funestus s.s. and allied species in southern Africa