Background: Tuberculosis, a communicable disease with significant morbidity and mortality, is the leading cause of death in the world from bacterial infectious disease. Because of its public health importance, there is need for rapid and definitive method of detecting the causative organism. Several approaches have been attempted, but the molecular methods, especially Polymerase Chain Reaction assays are the most promising for rapid detection of Mycobacterium tuberculosis complex from clinical samples. Aim: This study was aimed at using Polymerase Chain Reaction for detection of Mycobacterium tuberculosis complex from clinical samples using universal sample processing methodology. Subjects and Methods: Two hundred clinical samples sent to Tuberculosis laboratories in Ibadan and Osogbo, Nigeria, were enrolled in this study. The samples were processed by universal sample processing methodology for PCR; smear microscopy was carried out on sputum samples by Ziehl Nelseen staining technique; and cultured on Middlebrook agar medium containing oleic acid albumin dextrose complex supplement after decontamination of samples. Results: Ninety six (48%) samples were detected positive for M. tuberculosis complex by polymerase chain reaction using the combination of boiling and vortexing and microscopy detected 72 (36%) samples positive for acid fast bacilli. Using culture method as gold standard, it was found that polymerase chain reaction assay was more sensitive (75.5%) and specific (94.8%) than microscopy (sensitivity of 48.5% and specificity of 85.7%) in detecting M. tuberculosis complex from clinical samples. There was significant difference in detecting M. tuberculosis from clinical samples when compared to microscopy (p<0.05). Conclusion: The study recommends that direct molecular detection of M. tuberculosis complex is sensitive and specific and polymerase chain reaction method should be used as an adjunct to other methods of laboratory diagnosis of tuberculosis.