peer reviewed ; The Anopheles gambiae complex is the most important vector for malaria in Sub-Saharan Africa, besides to other vectors such as Anopheles funestus. Malaria vector control must consider specific identification, the genetic diversity and the population structure of An. gambiae to design vector control strategies. The aim of this study was to determine the distribution of sibling species of the An. gambiae complex according to climatic regions related to cotton-growing or cotton-free areas by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Secondly, variation in mitochondrial cytochrome c oxidase 1 (COI) was used to assess the genetic structure within and between populations from our selected ecological zones. At the sibling species level, in all samples (n = 180), the following proportions of An. coluzzii (65.56%), An. gambiae s.s. (21.11%) and An. arabiensis (3.33%) were found. Hybrids between An. gambiae s.s. and An. coluzzii (7.78%) and hybrids between An. coluzzii and An. arabiensis (2.22%) were found as well. The phylogenetic tree and Integer Neighbour-Joining (IntNJ) haplotype network revealed no distinct genetic structure pattern related to climatic or agricultural conditions in Burkina Faso. The Fst (Wright’s F-statistic) values close to zero showed a free gene flow and an absence of differentiation in An. gambiae complex populations. Furthermore, neutrality indices calculated by Tajima’s D, Fu and Li's D*, Fu and Li's F*, Fu’s Fs tests suggested an excess of rare mutations in the investigated populations. Overall, this study found variations in the proportions of An. gambiae s.s., An. coluzzii and An. arabiensis according to climatic regions but without population 35 structuration of the An. gambiae complex. These results are scientific contributions that can be used as a basis for further in-depth study of the genetic diversity of the An. gambiae complex for epidemiologic risk assessment of malaria in Burkina Faso.