We made the first analysis of the COI gene sequences of 22 species of spittlebugs and aquatic true bugs sampled in São Paulo State (Brazil) and used this information to determine the variability within these groups. Considering each codon position, we observed that the third base was the most variable, and the first base was the most conserved. Among species, Mahanarva fimbriolata and Deois flavopicta had the greatest genetic distance (0.181), and Notozulia entreriana and Mahanarva sp had the smallest distance (0.055), with an average variation of 0.119. In Gerromorpha, the greatest distance occurred between Halobatopsis platensis and Rhagovelia zela (0.401), while between Cylindrostethus palmaris and Brachymetra albinervis albinervis, the distance was only 0.187; the average value observed for the Gerromorpha was 0.265. In the Nepomorpha, the species Buenoa antigone antigone and Belostoma micantulum had the greatest genetic distance (0.337), while Martarega brasiliensis and B. a. antigone had the smallest (0.154). The average value observed for Nepomorpha was 0.203. In Cicadomorpha (Auchenorrhyncha) and Nepomorpha (Heteroptera), the COI gene has been conserved; however, it is still useful for characterization of the different taxa. COI analysis was unable to resolve some of the Gerromorpha groups.