Abstract Introduction The aim of this retrospective study was to determine the prevalence of three-rooted mandibular permanent first molars among the Indian population by using periapical radiographs. Methods Five hundred eighty-six patients (320 females and 266 males) were selected, with at least 1 mandibular first molar. A total of 1054 periapical radiographs of mandibular first molars, comprising 526 right side and 528 left side, were included. The radiographs were taken at 30-degree mesial angulation and were evaluated by using the magnifying lens. The incidence, gender, and symmetry of three-rooted mandibular first molars were recorded and analyzed by using the χ2 test. Results The prevalence of three-rooted mandibular first molars was 5.97% for all patients and 4.55% for all teeth, respectively. The incidence of bilateral symmetrical distribution was 37.14%. The incidence was 6.88% for female patients and 4.89% for male patients (χ2 = 1.02, P > .05) and 4.94% for the right side and 4.17% for the left side, respectively (χ2 = 0.36, P > .05). No statistically significant differences were found between female and male patients and between the right-side and left-side occurrences. Conclusions Clinicians should be aware of the high racial prevalence of this unusual root morphology in mandibular first molars among the Indian population before and during the root canal treatment of three-rooted mandibular first molars.