The PCR and its variations such as nested-PCR are currently considered as a rapid and sensitive method for detection of Mycobacterium tuberculosis. The standard laboratory procedure for diagnosing the tuberculosis (TB) disease based on microscopic examination of acid-fast bacilli has low sensitivity, and mycobacterium culture is time-consuming. In addition, in some cases obtaining samples for smear and culture is difficult. Urine may, therefore, be a convenient, noninvasive sample to use for the identification and diagnosis of M. tuberculosis. The current study aimed at evaluating the diagnostic value of nested-PCR to detect M. tuberculosis in the urine of patients with smear positive pulmonary TB. This case–control study included 60 patients with proven smear positive pulmonary TB (according to the National TB Protocol) and 30 patients who were completely healthy. DNA extraction and nested-PCR testing were performed on all urine samples. Result of the current study indicated that nested-PCR of urine for M. tuberculosis was positive in 30% (18/60) of the patients. The control group all had negative urine PCR (sensitivity 30% and specificity 100%). Because of the ease of urine sample preparation and the 100% specificity of the PCR and nested-PCR method, a urine sample could be used as a diagnostic aid in smear positive pulmonary TB cases, in which obtaining a sputum sample is problematic.