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Abstract BackgroundInsecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles ofAnopheles funestusandAnopheles arabiensiswere examined across mosquito populations from and within neighbouring villages. MethodsMosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed withAnophelesmosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure. ResultsPhenotypic insecticide resistance to deltamethrin was observed inAn. funestussensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% ofAn. funestus s.s.wereCYP6P9ahomozygous resistant.An. arabiensiswas phenotypically susceptible to deltamethrin and 99% werekdrhomozygous susceptible. BothAnophelesspecies exhibited high allelic richness and heterozygosity. Significant deviations from Hardy–Weinberg equilibrium were observed, and high linkage disequilibrium was seen forAn. funestus s.s.,supporting population subdivision. However, the F_STvalues were low for both anophelines (− 0.00457 to 0.04213), N_mvalues were high (9.4–71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, andStructureanalysis showed no clustering ofAn. funestus s.s.andAn. arabiensispopulations. These results suggest high gene flow among mosquito populations. ConclusionDespite a relatively high level of phenotypic variation in theAn. funestuspopulation, molecular analysis shows the population is admixed. These data indicate thatCYP6P9aresistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.