The aim of this study was to evaluate the value of Cobas Amplicor Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) system in the rapid diagnosis of tuberculosis. During the study period, the results of acid-fast stain (AFS), culture and Cobas Amplicor MTB system obtained from 937 clinical (158 respiratory and 779 non-respiratory) specimens were retrospectively evaluated. When culture results were accepted as the gold standard, the sensitivity, specificity, positive and negative predictive values of Cobas Amplicor MTB PCR system were found as 100% for smear-positive respiratory specimens, 75%, 91.7%, 40% and 98% for smear-negative respiratory specimens, and 89.5%, 91.8%, 65.4% and 98.1% for all of the respiratory specimens, respectively. These rates were found as follows for smear-positive, smear-negative and all of the non-respiratory specimens, respectively; 100%, 66.7%, 83.3% and 100%; 29.2%, 97.3%, 26.9% and 97.6%; and 50%, 97.1%, 44.7% and 97.6%. The overall inhibition rate for Cobas Amplicor MTB was 4.8%. In conclusion, Cobas Amplicor MTB PCR system was considered as a rapid and reliable method for the diagnosis of tuberculosis in smear positive pulmonary samples. However, in smear negative pulmonary samples and non-respiratory samples, test results should be evaluated together with clinical and other laboratory data.