Mycobacterium simiae is usually an environmental contaminant rarely associated with human disease. We report a fatal case of M.simiae infection in a 37 year old, HIV positive, male from whom the organism was isolated from blood culture. The identification of M.simiae was performed using DNA amplification followed by analysis on 3% agarose gel of the amplicon fragments after digestion by restriction endonucleases. The precise identification of mycobacterial isolates to the species level is important, with both epidemiological and therapeutic implications.