Background: Environmental changes resulting from the urbanization process represent a challenge for malaria control. The majority of malaria cases in South America occur in rural areas, areas of recent occupation, mining and indigenous areas of the Amazon region. Although these areas have a significant impact on malaria cases, few entomological studies have been carried out in areas of recent occupation. This study aimed to describe the density, natural infection rate and hematophagic behavior of anopheline species in two settlements in the state of Rondonia, Brazil in order to understand how malaria transmission occurs in areas that have been settled at different times.Methods: An area of recent occupation, denominated Acampamento Fortaleza (AF), and an old settlement, denominated Projeto de Assentamento Florestal Jequitibá (PAFJ), were studied. Peridomicile collections of anopheles were carried out using the Protected Human Attraction Technique (PHAT). The risk and potential for malaria transmission were assessed using the human biting rate (HBR), sporozoite rate (SR) and the entomological inoculation rate (EIR).Results: The results confirmed that Nyssorhynchus darlingi, the main vector responsible for the transmission of malaria in the state of Rondônia, is the predominant species in the two studied locations. Although settlement of the two study sites has occurred at different times, the species richness found was low, showing that the environmental changes caused by anthropological actions probably favor the adaptation of the Ny. darlingi species. Of the 615 anopheline mosquitoes assessed, 7 (1.1%) were positive for Plasmodium infections. The EIR revealed that Ny. darlingi contributes to the transmission of malaria in both locations, since it was responsible for 0.41 infectious bites in humans at night in PAFJ and 0.16 in AF. In the two study sites, the biting occurred more frequently at dusk.Conclusions: Ny. darlingi is the principal vector found in the studied locations. Its prevalence occurs in areas of recent colonization but, even when present in a low density, this species could maintain transmission of malaria in an older settlement. The entomological information obtained in this study is important and may aid the selection of vector control actions in these locations that are considered as having a high risk of malaria transmission.