Objective To evaluate the sensitivity and specificity of a nested polymersase chain reaction (nested PCR) for detection of the Mycobacterium tuberculosis IS6110 in paraffin embedded tissues. Methods 31 samples from tuberculosis patients and 5 biopsy specimens from patients with hepatitis were subjected to detection of Mycobacterium tuberculosis IS6110 by nested PCR. PCR products of two randomly selected samples were cloned and sequenced. Results Mycobacterium tuberculosis IS6110 was positive in 28 of 31 samples of tuberculosis. The sensitivity and specificity of nested PCR for detection of IS6110 were 90.3% and 100%, respectively. The predictive value of nested PCR was 100%. The sequences of two samples were compared with known sequence of H37Rv isolate (reported by Thierry D) and with nucleotide homology of 97% and 95.3%, respectively. Conclusions Nested PCR is sensitive and specific in the diagnosis of Mycobacterium tuberculosis infection in tissues of routinely paraffin embedded. We propose the diagnostic application of nested PCR for the identification of Mycobacterium tuberculosis infection, especially in the cases which can not be distinguished with certainty from other diseases by histopathology and Ziehl Neelsen's staining.