Objective: This study aimed to use the polymerase chain reaction (PCR) as a rapid tool for the diagnosis of pulmonary tuberculosis from sputum samples. Clinically suspected tuberculosis patient in Khartoum state were targeted. Materials and Methods: Sputum specimens were collected from patients attending AbuAngaHospital, Alsha'ab Teaching Hospital and Tuberculosis Reference Laboratory. Patients were consented and informed. Sputum samples were stained with ZN stain, then decontaminated and cultured on LJ medium. Part of the sputum was used for DNA extraction by isopropanol method for PCR. Results: 37(21.6%) smears from the collected 171 sputum samples showed positive ZN smears while 134 (78.4%) showed negative result. On LJ medium, 23.4% showed MTC-like colonies, 5.8% were considered rapidly growing Mycobacteria and 70.8% samples revealed contamination or no growth. The MTC-like colonies were confirmed by conventional methods. When PCR was performed, 142 (83%) samples showed a band typical in size (123 bp) to the target gene of Mycobacterium tuberculosis complex (IS 6110) as indicated by the standard DNA marker. 29(17%) samples were negative. Conclusion: These results revealed clearly the importance, feasibility and sensitivity of PCR as a rapid diagnostic tool to detect M. tuberculosis directly from sputum samples.