Background This study was aimed at establishing age estimates based on distal root development of the mandibular third molar for Ugandans aged 10–22 years. This was a cross-sectional study using orthopantomograms (OPGs) of 671 patients attending the Mulago Hospital Dental Clinic. The patients’ chronological age and sex were obtained from either their national identity cards or birth certificates (females; n = 326, 48.6%). Third molar root development was assessed using Demirjian, Goldstein, and Tanner (DGT), modified Demirjian by Solari, Moorrees, Fanning, and Hunt (MFH), and Haavikko methods. Age was summarized using means/standard deviation (SD), medians, and lower and upper quartiles. Sex differences were assessed using Student’s t-test. Results Crown completion (stage D) and root initiation (stage Ri) were observed at 12.6 years for females and 13.5 years for males (P = 0.02), while complete apex closure occurred at 19.8 for females and 20.1 for males (P = 0.3). There were statistically significant differences in Demirjian root stages E, F, and G and MFH and Haavikko stages Ri, R1/4, and R1/2 between the sexes (P < 0.05). The difference in the mean age of root development between females and males ranged between 0.9 year at DGT/MFH root stages D and Ri (13.5–12.6) and 1.4 years at Solari, MFH, and Haavikko root stages F, R1/4, and R1/2 (16.3–14.9, 17.1–15.7). No differences were observed between the right and left mandibular molars. Conclusions This study provides age reference standards based on third molar root development specific to the Ugandan adolescent and young adult population. The findings can be used to formulate contemporary standards and utilized as reference material to assess third molar maturity for forensic purposes.