Prostate cancer (PC) is one of the most common cancers and the second leading cause of mortality from cancer in Colombian men. Mitochondrial DNA (mtDNA) haplogroups have been associated with the risk of PC. Several studies have demonstrated dramatic differences regarding the risk of PC among men from different ethnic backgrounds. The present study was aimed at assessing the relationship between mtDNA haplogroups and PC. The mitochondrial DNA hypervariable segment I (HSV-1) was sequenced in a population-based study covering 168 cases (CA) and 140 unrelated healthy individuals as a control group (CG). A total of 92 different mtDNA sequences were found in CA and 59 were found in the CG. According to the geographical origin attributed to each mtDNA haplogroup, 82% of the mtDNA sequences found in both groups were Native Americans (A, B, C, and D). The most frequent was A (41.1%CA-42.1%CG), followed by B (22.0%CA-21.4%CG), C (12.0%CA-11.4%CG), and D (6%CA-10.0%CG). A lower percentage of European haplogroups (U, H, K, J, M, T, and HV) were also found (13.1%CA-12.9%CG), likewise African haplogroups (L0, L1, L2, and L3) (6.5%CA-2.1%CG). There were no statistically significant differences between the distribution of mtDNA haplogroups in CA and the CG in this study.