Primary prostate tumor heterogeneity is poorly understood, leaving research efforts with challenges regarding the initiation and advancement of the disease. The growth of tumor cells is accompanied by mutations in nuclear and in mitochondrial genomes. Thus, mitochondrial DNA mutations may be used as tumor cell markers. By the use of laser capture microdissection coupled with assays for mitochondrial point mutation detection, mtDNA mutations were used to trace mutated cells at a histological level. Point mutations in mtDNA were determined in 12 primary prostate cancers. The tumors represent different pathology-prognostic grade groups. Known mutational hotspots of the mtDNA were scanned for heteroplasmy. All specimens with mtDNA heteroplasmy were subsequently subsampled by laser capture microdissection. From a total number of 1728 microsamples, mitochondrial DNA target sequences were amplified and base substitutions detected by cycling temperature capillary electrophoresis. Real-time PCR was used as a quantitative assay to determine the relative mtDNA copy number of 12 tumors studied, represented by two samples from each (N = 24); a high degree (75%) demonstrated tumor specimen heterogeneity. A grid of 96 spots isolated by laser capture microdissection demonstrated interfocal sample heterogeneity and increased the limit of detection. The spots demonstrated a wide range of mutant fractions from 0 to 100% mutant copies. The mitochondrial DNA copy number in the samples was determined by real-time PCR. No correlation between copy number and pathology-prognostic grade groups was observed. Somatic mitochondrial DNA point mutations represent traceable biomarkers demonstrating heterogeneity in primary prostate cancer. Mutations can be detected in areas before changes in tissue histopathology are evident to the pathologist.