The purpose of this study was to explore the speaker-discriminatory potential of vowel formant mean frequencies in comparisons of identical twin pairs and non-genetically related speakers. The influences of lexical stress and the vowels’ acoustic distances on the discriminatory patterns of formant frequencies were also assessed. Acoustic extraction and analysis of the first four speech formants F1-F4 were carried out using spontaneous speech materials. The recordings comprise telephone conversations between identical twin pairs while being directly recorded through high-quality microphones. The subjects were 20 male adult speakers of Brazilian Portuguese (BP), aged between 19 and 35. As for comparisons, stressed and unstressed oral vowels of BP were segmented and transcribed manually in the Praat software. F1-F4 formant estimates were automatically extracted from the middle points of each labeled vowel. Formant values were represented in both Hertz and Bark. Comparisons within identical twin pairs using the Bark scale were performed to verify whether the measured differences would be potentially significant when following a psychoacoustic criterion. The results revealed consistent patterns regarding the comparison of low-frequency and high-frequency formants in twin pairs and non-genetically related speakers, with high-frequency formants displaying a greater speaker-discriminatory power compared to low-frequency formants. Among all formants, F4 seemed to display the highest discriminatory potential within identical twin pairs, followed by F3. As for non-genetically related speakers, both F3 and F4 displayed a similar high discriminatory potential. Regarding vowel quality, the central vowel /a/ was found to be the most speaker-discriminatory segment, followed by front vowels. Moreover, stressed vowels displayed a higher inter-speaker discrimination than unstressed vowels in both groups; however, the combination of stressed and unstressed vowels was found even more explanatory in terms of the observed differences. Although identical twins displayed a higher phonetic similarity, they were not found phonetically identical.