Anopheles species identification is relevant for malaria vector research and to direct control strategies, but difficulties arise among closely related species when using traditional taxonomy. To solve this problem, molecular markers such as the ribosomal internal transcribed spacer 2 (ITS2), have been proposed for species confirmation and taxonomy. Previous studies suggested that at least one Compensatory Base Change (CBC) in the ITS2 secondary structure between lineages could be an indication of different biological species. In the current study, a comparison of the ITS2 secondary structure for five morphologically closely related species that are potential malaria vectors in Colombia was carried out. The ITS2 secondary structure for Anopheles (Anopheles) calderoni was predicted and used as template for homology modeling of ITS2 for Anopheles punctimacula s.s., Anopheles malefactor, Anopheles neomaculipalpus and Anopheles apicimacula. Conserved motifs, CBCs and a distance-based phylogeny were inferred using multiple sequence alignments and secondary structure comparisons. Overall, a typical ITS2 structure with four helices, a mismatch in helix II (C-U) and an AAA motif between helices II and III, was detected. Comparisons among species revealed one to four CBCs, mainly located in helices II and III. The ITS2 structure of An. punctimacula s.s., An. malefactor and An. calderoni were the closest compared with An. neomaculipalpus and An. apicimacula, confirming previous phylogenetic relationships. This preliminary work demonstrates that ITS2 secondary structure analysis has potential for distinguishing among closely related Anopheles species.