The early differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria (NTM) and the identification of NTM species are crucial for the proper management of patients with smear-positive sputum. We evaluated the usefulness of a polymerase chain reaction restriction analysis (PRA) method based on the rpoB gene for identifying NTM species in a study of 121 smear-positive respiratory specimens with presumed NTM. The PRA method amplified mycobacterial DNA in 72 specimens (60%) and differentiated NTM species correctly in 68 (94%). The PRA method could be a useful and rapid method for identifying NTM species in smear-positive respiratory specimens when urgent clinical decisions are required.