This review is to summarize recent developments in the field of mycobacteriology since the diagnosis of tuberculosis remains elusive in spite of our best efforts and great scientific advances. Progress has been made in further improving upon the age old, time tested traditional techniques like microscopy (Auramine-Rhodamine Fluorescent staining and peptide nucleic acids), culture and sensitivity techniques (solid, liquid, radiometric, and non-radiometric systems) that still remain as the gold standard for its diagnosis. Development of rapid methods [(high performance liquid chromatography, thin layer chromatography, RNA sequencing and polymerase chain reaction (PCR), nucleic acid sequence based amplification assay (NASBA), Transcription mediated assay (TMA) and Ligase chain reaction (LCR)] have paved the way for its rapid detection and treatment. It is interesting to see the role of molecular assays appearing more often in literature now. The molecular amplification systems (PCR, NASBA, TMA, LCR) besides identifying Mycobacterium tuberculosis (MTB) as well as non-tuberculous mycobacteria (NTM), directly from the sample can also identify Rifampin (rpoB gene)/Isoniazide (katG gene) resistant strain. Molecular assays have been found useful particularly in smear positive sputum with high sensitivity and specificity whereas variable sensitivity for sputum negative and extra pulmonary specimens has been observed. Representative specimen and its quality affect the performance of these assays. Emphasis should given to proper collection and transportation of the representative specimen for appropriate evaluation.