Anopheles darlingi is a major vector for malaria in Central and South America. Behavioral, ecological, genetic, and morphologic variability has been observed across its wide distribution. Recent studies have documented that 2 distinct genotypes exist for An. darlingi: a northern lineage (Belize, Guatemala, Colombia, Venezuela, and Panama) and a southern lineage (Amazonia and southern Brazil). In order to determine if these genotypes exhibited different behavioral traits, entrance and exit movement patterns between 2 field populations of An. darlingi that represented each genotype were evaluated using experimental huts. The Belize population exhibited bimodal entrance, with peak entry occurring between 7:00–8:00 p.m. and 5:00–6:00 a.m. and peak exiting occurring between 7:00–8:00 p.m. The Peru population exhibited unimodal entrance, with peak entry occurring between 10:00–11:00 p.m. and peak exiting occurring between 11:00–12:00 a.m. with a secondary smaller peak at 2:30 a.m. Entrance and exit behavioral patterns were significantly different between the Belize and Peru populations of An. darlingi (log-rank [Mantel–Cox] P < 0.001). Information from the present study will be used in the future to determine if there is a correlation between genotype and host-seeking behavior and can be used in the present for regional vector risk assessment.