Introduction: Knowing the morphology of the pulp chamber and root canals, as well as their diversity, is key to endodontic treatment success. Mandibular molars are a group of teeth with complex internal anatomy of which knowledge is paramount. Objective: The objective of this study was to assess, by means of four methods (radiographic, macroscopic, microscopic and anatomical), the amount and configuration of root canals present in mandibular first molars. Methods: This was a cross-sectional ex vivo, observational and descriptive study conducted with 50 permanent mandibular first molars. The following exclusion criteria were applied: teeth with completely destroyed crowns, incomplete root formation, resorption and/or perforation, including healthy and semi-healthy crowns. Results: A total of 24% mandibular first molars had three root canals, with a cross-section flattened mesiodistally and elongated buccolingually to the full extent of mesial (48.6%) and distal canals (52.2%) being the most prevalent. However, other possible configurations, such as circular morphology both in mesial (46.6%) and distal canals (38.1%), and cases of five root canals (16%) could also be seen. Conclusion: The methods adopted allowed us to find that, in mandibular first molars, there was a prevalence of root canals flattened mesiolingually and elongated buccolingually. Three root canals were prevalent; however, variations may occur.