Tuberculosis, is a life-threatening infectious disease and one of the leading causes of mortality worldwide. It is a multisystemic disease, and involvement of lungs and pleura is seen in majority of patients. The global control of tuberculosis is impeded by the relatively low sensitive conventional diagnostic assays, especially for drug resistant strains. The current gold standard for tuberculosis diagnosis is the combination of culture and clinical diagnosis. Since conventional diagnostic assays require long time, are labor intensive and insufficient for species-level identification, new solutions for problems in routine diagnostic applications for tuberculosis are required. Implementation of reliable, highly specific and sensitive assays which were standardized for rapid diagnosis of Mycobacterium tuberculosis, is recommended by Centers for Disease Control and Prevention (CDC). Implementation of nucleic-acid amplification based tests (NATs) to support rapid diagnosis especially in reference laboratories or health-care clinical laboratories, which use the diagnostic algorithms denoted in CDC guidelines, meet American Thoracic Society class 2 and 3 standards and have smear positive sample counts ≥ 500 per year, is accepted as a logical and cost-effective approach. Analysis of cost-effectiveness for NATs are also required for global TB management plans. All current meta-analysis on NAT for tuberculosis indicate that in-house assays display considerable variations in sensitivity and specificity and exhibit discrepancies with commercial NATs. Evaluations also notified that the specificity for NAT is usually higher than expected in laboratory diagnosis of both forms of tuberculosis, whereas sensitivity is very low with considerable inter-assay variations. In conclusion, it is thought that current NATs are not reliable as single assays for rapid tuberculosis diagnosis for routine testing and should not be used in screening, but they are valuable methods in supporting conventional assays for clinical follow-up of patients. This review article discusses the diagnostic value of molecular methods in the evaluation of pulmonary and extrapulmonary tuberculosis in the light of the current literature.