BACKGROUNDMalaria is a public health concern in the Amazonian Region, whereAnopheles darlingiis the main vector ofPlasmodiumspp. Several studies hypothesised the existence of cryptic species inAn. darlingi, considering variations in behaviour, morphological and genetic aspects. Determining their overall genetic background for vector competence, insecticide resistance, and other elements is essential to better guide strategies for malaria control.OBJECTIVESThis study aimed to evaluate the molecular diversity in genes related to behaviour and insecticide resistance, estimating genetic differentiation inAn. darlingipopulations from Amazonian localities in Brazil and Pacific Colombian region.METHODSWe amplified, cloned and sequenced fragments of genes related to behaviour:timeless(tim) andperiod(per), and to insecticide resistance: voltage-gated sodium channel (NaV) and acetylcholinesterase (ace-1) from 516An. darlingiDNA samples from Manaus, Unini River, Jaú River and Porto Velho - Brazil, and Chocó - Colombia. We discriminated single nucleotide polymorphisms (SNPs), determined haplotypes and evaluate the phylogenetic relationship among the populations.FINDINGSThe genesper,timandace-1were more polymorphic thanNaV. The classicalkdrandace-1Rmutations were not observed. Phylogenetic analyses suggested a significant differentiation betweenAn. darlingipopulations from Brazil and Colombia, except for theNaVgene. There was a geographic differentiation within Brazilian populations consideringperandace-1.CONCLUSIONSOur results add genetic data to the discussion about polymorphisms at population levels inAn. darlingi. The search for insecticide resistance-related mechanisms should be extended to more populations, especially from localities with a vector control failure scenario.