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• An. Aquasalis mosquitoes of a 25 year-established colony keep two phenotypes (30BS and 50BS) based on their 2nd hind-tarsomere scaling patterns. • Transcribed Spacer 2 (ITS2) confirm the species status and the phenotypes regardless of gender. • Male-oriented biases in both phenotypic categories were observed among the 28 genotypes. • Evaluating the infection of An. aquasalis to Plasmodium parasites, there was a higher susceptibility of 30BS compared with 50BS. Anopheles aquasalis is an important malaria vector in coastal regions of South America and islands of the Caribbean. In its original description, the species was divided into two varieties, based on the scaling patterns of their hind-tarsomere 2. Specimens from our 25-year established colony, used for Plasmodium experimental infections, still exhibit both scaling tarsomere patterns. This study examined the DNA sequence of the nuclear Internal Transcribed Spacer 2 (ITS2) and susceptibility to Plasmodium , looking for differences among the phenotypes 30BS and 50BS. One hundred mosquitoes, 25 males and 25 females of each sex, and phenotype were analyzed. Twenty-seven novel haplotypes were identified. Three were found in both phenotypes (30BS and 50BS) regardless of gender. Among the other 27 genotypes, we observed a male-oriented bias in both phenotypic categories. Evaluation of Plasmodium yoelii N67 infections, based on oocyst counts, showed a higher susceptibility of 30BS compared with 50BS. Future studies need to be conducted to evaluate if these genotype assortments among the phenotypic groups reflect differences in fitness, mating, and their susceptibility to infection by Plasmodium parasites. [ABSTRACT FROM AUTHOR]