Objectives: To evaluate the efficacy of nested polymerase chain reaction (PCR) with first void urine (FVU) for the diagnosis of Mycoplasma hominis in male patients.Methods: Matched FVU specimens and urethral swabs were collected from 194 male patients with Nongonococcal Urethritis and tested by nested PCR and cell culture. Cell culture was used as a gold standard for evaluating other assay techniques.Results: For FVU nested PCR assay and FVU cell culture,our results showed that the sensitivity was 100% and 93.3%;specificity was 97.0% and 98.2%; positive predictive value (PPV) was 85.7% and 90.3%, negative predictive value (NPV)was 100% and 98.8%, respectively. The total consistency between the two techniques was 97.4 %.Conclusions: For the diagnosis of Mycoplasma hominis in men, nested PCR detecting FVU is a highly sensitive and specific method. First void urine can replace swab culture or PCR in terms of acceptability and feasibility.