In this study mitochondrial D-Loop variations in Iranian prostate cancer and benign prostatic hyperplasia (BPH) patients were investigated. Tumour samples and corresponding non-cancerous prostate tissue from 40 prostate cancer patients and 40 age-matched BPH patients were collected. The entire mtD-loop region (16024-576) was amplified using the PCR method and products were gel-purified and subjected to direct nucleotide sequencing. A total of 129 variations were found, the most frequent being 263A${ ightarrow}$G and 310T${ ightarrow}$C among both BPH and prostate cancer patients. Variation of 309 C${ ightarrow}$T was significantly more frequent in prostate cancer patients (P value<0.05). Four novel variations were observed on comparison with the MITOMAP database. Novel variations were np16154delT, np366G${ ightarrow}$A, np389G${ ightarrow}$A and 56insT. There was no correspondence between the different variations and the age of subjects. Considering that D-loop variations were frequent in both BPH and prostate cancer patients in our study, the fact that both groups had high average age can be a possible contributing factor. D-loop polymorphisms and microsatellite instability can influence cell physiology and result in a benign or malignant phenotype. Significantly higher frequency of 309 C${ ightarrow}$T variation in cancer patients is a notable finding and must be a focus of attention in future studies.