Abstract: The present invention relates to a method for specifically detecting Mycobacterium tuberculosis and nontuberculous mycobacteria by simultaneously amplifying and analyzing target genes using various primers and probes, and a kit using same. The method of the present invention is capable of selectively detecting Mycobacterium tuberculosis and nontuberculous mycobacteria with very high efficiency through a multiplex real-time polymerase chain reaction (PCR) using probes and primers specific to target genes (particularly, IS6110, 16S rRNA and ?-actin). Also, the kit of the present invention is capable of conveniently and efficiently detecting the target genes in a sample through a multiplex real-time PCR. Therefore, the method and the kit of the present invention are capable of selectively detecting with ease whether or not there is an infection with Mycobacterium tuberculosis or nontuberculous mycobacteria in a sample, and can be more accurately applied to the treatment of diseases on the basis thereof.