Evaluation of a PCR-RFLP-ITS2 assay for discrimination of Anopheles species in northern and western Colombia

► We evaluate a PCR-RFLP-ITS2 assay for anopheline discrimination in Colombia. ► Stability of PCR-RFLP patterns should be evaluated since the ITS2 region could vary. ► ITS2 patterns in Anopheles species were conserved through the localities sampled. ► The assay discriminated species within the Oswaldoi Group and the Arribalzagia Series. ► The assay proved valuable for differentiating species with overlapping morphology.