Several lines of evidence suggest that loss of heterozygosity (LOH) in specific chromosomal regions is a common mechanism for the inactivation of tumor suppressor genes that are implicated in pathogenesis of prostate cancer (PCa). Short tandem repeat (STR) sequences are extremely reliable genetic markers for detection of LOH associated with cancers. Hence, in the current study, we investigated the detection of LOH at six STR markers (D8S360, D9S1748, D9S171, D8S137, D6S1631, and THRB) using blood circulating cell-free DNA (cfDNA), which can be used to distinguish PCa from benign prostatic hyperplasia (BPH).A total of 136 individuals were included in the study, 76 males diagnosed with PCa (50 males with localized PCa (LPCa) and 26 males with metastatic PCa (MPCa)) as experimental subjects and 60 males with BPH as controls. Circulating cfDNA was extracted from plasma samples and amplified with fluorescence-labeled primers specific for known STR markers. We also evaluated the serum prostate-specific antigen (PSA) in both groups.Our findings revealed that the frequency of LOH at D8S360, D9S1748, D9S171, D8S137, and D6S1631 was significantly higher in PCa subjects than in controls (p< 0.05). Of the six STR markers, LOH at D8S360 could discriminate MPCa from LPCa. We found that 71.05% of patients with PCa and 1.66%of BPH subjects had LOH at least at three of the markers in cfDNA.Our findings provide additional evidence to support the hypothesis that analysis of LOH at D8S360, D9S1748, D9S171, D8S137, and D6S1631 STR markers using cfDNA can be applied as a non-invasive diagnostic approach for the detection of PCa.