Abstract: Pleural fluid samples from patients with exudative effusion who were diagnosed with tuberculous pleuritis are examined using a new designed primer set based on IS1081 gene (IS1081-PCR) and rpoB-PCR. The PCR results are compared with the results of the sample cultures, using Loewenstein-Jensen (LJ) medium and Ziehl-Neelsen (ZN) staining. Of 78 cases that were confirmed as tuberculous pleuritis by histopathology, supported by sputum culture, biochemical markers (adenosine deaminase, gamma interferon and tumor necrosis factor), radiographic and clinical data, 61 (78.2%) were positive by IS1081-PCR, 43 (55.1%) by rpoB-PCR, 17 (21.7%) by culture and 3 (3.8%) by ZN stain. When IS1081-PCR test results were compared with the confirmed culture, the sensitivity, specificity, positive predictive value and negative predictive value for the IS1081-PCR were 94.1, 55.7, 37.2 and 97.1%, respectively. The corresponding values for the rpoB-PCR were 94.1, 26.2, 26.2 and 94.1%, respectively. When tests results were compare with the confirmed radiographic, histopathology, biochemical markers and clinical diagnosis of tuberculous pleuritis, the IS1081-PCR assay is more sensitive, specific and reliable than both rpoB-PCR assay and culture.