This study is the first one done for isolating and identifying both non-tuberculosis mycobacteria (NTM) and Mycobacterium tuberculosis (MTB) among tuberculosis suspected patients in Iraq, it based on combining conventional and molecular biology based characterization along with the examination of patterns of antimicrobial susceptibilities for using rpoB gene sequences by D-PCR proved to be a sensitive, specific and rapid technique to differentiate between MTB and NTM which can be used in Iraqi health centers to help treat and control these pathogens.