Objective: To evaluate and compare several anatomical parameters of mandibular first premolars from individuals from different Latin American countries using micro-computed tomography. Design: Five hundred extracted mandibular first premolars from Brazilian, Argentinian, Chilean, Colombian, and Ecuadorian individuals were scanned using micro-computed tomography (n = 100 teeth/country). Root canal configurations were classified according to established parameters. Analyses also included: canal volume and surface area, structure model index, distances from the apical foramen to the root apex or the cementoenamel junction, major/minor apical canal diameters, canal orifice shape, and prevalence of ramifications. Results: A single root was the most common anatomy in all countries (range, 97%−100%). Vertucci's type-I canal was the most frequent configuration (range, 36%−66%), followed by C-shaped and type-V canals. The oval-shaped canal orifice was the most predominant in all countries (range, 34%−58%), followed by the circular shape (range, 16%−47%). C-shaped canals occurred in all subpopulations (range, 14%−26%), always associated with radicular grooves. Ranges for canal ramifications were as follows: accessory canals, 36%−73%; lateral canals, 4%−12%; and apical delta, 4%−14% of the teeth. Many anatomic parameters differed significantly between countries (P < .05). Conclusions: Vertucci's types-I and -V, and C-shaped canals were the most prevalent configurations in the subpopulations investigated. Accessory canals and several complex anatomies were found, with some significantly different frequencies between countries.