The present invention relates to a multiplex PCR primer set capable of specifically amplifying a gene specifically existing in a Beijing family of Mycobacterium tuberculosis, and a gene capable of simultaneously diagnosing Mycobacterium tuberculosis and non-tuberculous mycobacteria, a composition and a kit comprising the primer set for simultaneous detection and discrimination of Mycobacterium tuberculosis and non-tuberculous mycobacteria, and a method for simultaneously detecting and discriminating Mycobacterium tuberculosis and non-tuberculous mycobacteria, comprising the primer set. Use of the primer set for simultaneous detection and discrimination of Mycobacterium tuberculosis and non-tuberculous mycobacteria of the present invention shows the effect of allowing for the effective discrimination of a Beijing family of Mycobacterium tuberculosis pandemic, with the concomitant discrimination and identification of clinically significant non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis (MTBC).