Five Anopheles marajoara Galvão and Damasceno populations, representing diverse ecological conditions, were sampled throughout Colombia and analyzed using nine hypervariable DNA microsatellite loci. The overall genetic diversity ( H = 0.58) was lower than that determined for some Brazilian populations using the same markers. The Caquetá population (Colombia) had the lowest gene diversity ( H = 0.48), and it was the only population at Hardy–Weinberg equilibrium. Hardy–Weinberg disequilibrium in the remaining four populations was probably caused by the Wahlund effect. The assignment analyses showed two incompletely isolated gene pools separated by the Eastern Andean cordillera. However, other possible geographical barriers (rivers and other mountains) did not play any role in the moderate genetic heterogeneity found among these populations ( F ST = 0.069). These results are noteworthy, because this species is a putative malaria vector in Colombia.