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Abstract Background The colonization of mosquitoes susceptible to Plasmodium vivax via direct membrane feeding assay (DMFA) has the potential to significantly advance our knowledge of P. vivax biology, vector-parasite interaction and transmission-blocking vaccine research. Anopheles darlingi and Anopheles deaneorum are important vectors of malaria in the Western Brazilian Amazon. Since 2018, well-established colonies of these species have been maintained in order to mass produce mosquitoes destined for P. vivax infection. Plasmodium susceptibility was confirmed when the colonies were established, but susceptibility needs to be maintained for these colonies to remain good models for pathogen transmission. Thus, the susceptibility was assessed of colonized mosquitoes to P. vivax isolates circulating in the Western Amazon. Methods Laboratory-reared mosquitoes from F10-F25 generations were fed on P. vivax blood isolates via DMFA. Susceptibility was determined by prevalence and intensity of infection as represented by oocyst load seven days after blood feeding, and sporozoite load 14 days after blood feeding. The effect of infection on mosquito survival was evaluated from initial blood feeding until sporogonic development and survival rates were compared between mosquitoes fed on infected and uninfected blood. Correlation was calculated between gametocytaemia and prevalence/intensity of infection, and between oocyst and sporozoite load. Results Significant differences were found in prevalence and intensity of infection between species. Anopheles darlingi showed a higher proportion of infected mosquitoes and higher oocyst and sporozoite intensity than An. deaneorum. Survival analysis showed that An. deaneorum survival decreased drastically until 14 days post infection (dpi). Plasmodium vivax infection decreased survival in both species relative to uninfected mosquitoes. No correlation was observed between gametocytaemia and prevalence/intensity of infection, but oocyst and sporozoite load had a moderate to strong correlation. Conclusions Colonized An. darlingi make excellent subjects for modelling pathogen transmission. On the other hand, An. deaneorum could serve as a model for immunity studies due the low susceptibility under current colonized conditions. In the application of DMFA, gametocyte density is not a reliable parameter for predicting mosquito infection by P. vivax, but oocyst intensity should be used to schedule sporozoite experiments.