Age estimation is a major step in forensic and legal procedures. Its relevance has been increasing due to growing society issues, such as identification of missing people, crimes against minors or lack of valid identification papers from locals or foreigners. Evaluation of the cut-off value of the Third Molar Maturation Index (I3M) = 0.08 for discriminating minors from adults in the Portuguese population. The left lower third molars were analysed by applying a specific cut-off value of 0.08 determined by Cameriere et al. in 2008. A sample of 778 digital panoramic radiographs of a representative Portuguese sample (442 females and 336 males), in the age range of 12–24 years (mean age 17.7 ± 2.98 years in females and 18.1 ± 3.0 years in males), was retrospectively evaluated. I3M decreased as the real age gradually increased in both sexes. The 0.08 cut-off score was valuable in discriminating adults from minors. According to the pooled results, the accuracy, by means of area under the curve, was 92.8% (95% confidence interval (CI) 91.0–94.6%). The proportion of correctly classified subjects (sensitivity) was 90.7% (95% CI 88.7–92.8%) and the specificity was 94.9% (95% CI 93.3–96.4%). The results show that I3M is a valuable method to differentiate minors from adults in the Portuguese population.