Mycobacteria comprise a diverse group of bacteria that are widespread in nature, some of which cause significant disease in humans. Members of the Mycobacterium tuberculosis complex (MTBC) are the most important human pathogens of the genus Mycobacterium. Traditional methods for detection and identification of mycobacteria include microscopy, culture and phenotypic tests. These methods either lack sensitivity, specificity, or are time consuming. Advances in the field of molecular biology have provided rapid diagnostic tools that have reduced the turnaround times for detecting MTBC and drug resistance in cultures and directly in clinical specimens from weeks to days. This review discusses the molecular genetic techniques for detecting and identifying MTBC as well as drug resistance of mycobacteria in clinical specimens.