The study underpins barcode characterization of insect species collected from Saudi Arabia and explored functional constraints during evolution at the DNA and protein levels to expect the possible mechanisms of protein evolution in insects. Codon structure designated AT-biased insect barcode of the cytochrome C oxidase I (COI). In addition, the predicted 3D structure of COI protein indicated tyrosine in close proximity with the heme ligand, depicted substitution to phenylalanine in two Hymenopteran species. This change resulted in the loss of chemical bonding with the heme ligand. The estimated nucleotide substitution matrices in insect COI barcode generally showed a higher probability of transversion compared with the transition. Computations of codon-by-codon nonsynonymous substitutions in Hymenopteran and Hemipteran species indicated that almost half of the codons are under positive evolution. Nevertheless, codons of COI barcode of Coleoptera, Lepidoptera and Diptera are mostly under purifying selection. The results reinforce that codons in helices 2, 5 and 6 and those in loops 2-3 and 5-6 are mostly conserved and approach strong purifying selection. The overall results argue the possible evolutionary position of Hymenopteran species among those of other insects.