BackgroundInformation on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vectorAnopheles coluzziifrom Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon.ResultsGenome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies—including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), theacetylcholinesterase-1 gene (G280S) and theCYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony ofAn. coluzzii(Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes,GSTe2andCYP6Z2, were functionally validated. TransgenicDrosophila melanogasterflies expressingGSTe2exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5′ intergenic region, to identify putative nucleotide(s) associated withGSTe2overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression ofGSTe2in the resistant mosquitoes. Transgenic flies expressingCYP6Z2exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed inCYP6Z2transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations ofAn. coluzziioverexpressing this P450.ConclusionsThese findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12915-023-01610-5.