The increasing frequency of multidrug-resistant strains of M. tuberculosis becomes dramatic in industrialized countries as well as in developing countries, particularly among patients infected with human immunodeficiency virus. It needs to formulate rapid strategies for diagnosing multidrug-resistant tuberculosis. For these new drug resistance, novel detection methods are developed in order to identify the resistant strains and to undertake efficacious antituberculosis therapies more rapidly. The phenotypic methods are based on the measurement of the microbial growth on nutritional supplement with antimicrobial agents; however, these proportional methods, such as the method in solid medium, the Bactec radiometric method or the MGIT method (mycobacterial growth indicator tube), are time consuming and give results in 5 to 21 days. In contrast, the genotypic tests, using knowledge of the genes involved in the resistance, reduce the time to detection of resistance from weeks to days. After amplification of the segment of the gene encoding the drug target by PCR, these methods are based on the identification of the different mutations conferring the antimicrobial resistance in M. tuberculosis. These methods are applied with success for detection of rifampicin resistance, conferring by mutations in a defined region of the rpoB gene for 99% of cases; on the contrary, results are less for other antituberculous drugs because of the insufficiency of knowledge of the molecular basis of drug resistance.