BACKGROUND. Malaria is a public health concern in the Amazonian region, where Anopheles darlingi is the main vector of Plasmodium spp. Several studies hypothesized the existence of cryptic species in An. 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. OBJECTIVES. This study aimed to evaluate the molecular diversity in genes related to behaviour and insecticide resistance, estimating genetic differentiation in An. darlingi populations from Amazonian localities in Brazil and Pacific Colombian region. METHODS. We amplified, cloned and sequenced fragments of genes related to behaviour: timeless (tim) and period (per), and to insecticide resistance: voltage-gated sodium channel (NaV) and acetylcholinesterase (ace-1) from 516 An. darlingi DNA samples from Manaus, Unini River, Jaú River and Porto Velho – Brazil, and Chocó – Colombia. We discriminated SNPs, determined haplotypes and evaluate the phylogenetic relationship among the populations. FINDINGS. The genes per, tim and ace-1 were more polymorphic than NaV. The classical kdr and ace-1R mutations were not observed. Phylogenetic analyses suggested a significant differentiation between An. darlingi populations from Brazil and Colombia, except for the NaV gene. There was a geographic differentiation within Brazilian populations considering per and ace-1. CONCLUSIONS. Our results add genetic data to the discussion about polymorphisms at population levels in An. darlingi. The search for insecticide resistance-related mechanisms should be extended to more populations, especially from localities with a vector control failure scenario.