Mitochondrial genomes (mtDNA) were analyzed to find polymorphic segments: neutral (N), slightly neutral (SN), selective (S) or slightly selective (SS) with the aim to increase the power of discrimination in DNA identification process. For this purpose, we developed an algorithm supported by three indexes to analyze 3295 mtDNA (47 populations). We obtained 576 polymorphic segments in mtDNA using limits without polymorphism ≥6 nucleotides. The SN and N were frequent in the electron transport chain-complex and larger than SS and S (mean 104pb vs 54pb). The multivariate analysis showed a differential structure among segments using the combined segment index (nMDS stress = 0.07 and ANOSIM R = 42). Therefore, the SN and N could be applied for identification and the SS and S for phenotypic association as an additional tool in DNA identification process.