This study was conducted to investigate the root canal anatomy of maxillary first molars in Black South Africans. Micro-computed tomography was used to investigate 101 maxillary first molars (53 teeth from the right, 48 from the left; 50 male and 51 female teeth). The number of root canals in each tooth was determined, and the relationship between side, sex and age was analyzed using chi-squared test. To determine intra- and inter-observer reliability, Cohen's kappa coefficients were calculated. Intra- and inter-rater agreements of 96.92% and 98.08% were achieved, respectively. Most teeth contained either three or four canals, but a second, third and fourth mesio-buccal canal was found in 60.39%, 5.94% and 0.99% of teeth, respectively. The disto-buccal and palatal roots contained predominantly single canals, but additional canals were noted in 2.97% and 1.98% of teeth. Four canals were common in females and teeth on the right side often contained a second mesio-buccal canal. However, the prevalence of a third mesio-buccal canal was higher in males than in females. The teeth studied showed diversity and variations between sexes and arch sides. These findings will aid clinicians in endodontic treatment and will be applicable for educational purposes.