Tuberculosis (TB) is one of the most important AIDS associated infectious diseases worldwide. It is a leading cause of illness and death among people with HIV/AIDS in resource-poor areas of the world. The annual incidence of TB among indigenous Iranians stands at 14 cases per 100,000 inhabitants. This study aimed to identify Mycobacterium infection among Iranian HIV positive patients. Two sputum specimens were collected from smear positive AIDS patients. Samples were cultured on Lowenstein-Jensen media for three weeks. DNA was extracted from two samples based on van Embden protocol. To identify Mycobacterium tuberculosis complex, a PCR was conducted to amplify a 245 bp fragment of IS6110 element, followed by RD12 method to confirm PCR result. Whole DNARFLP with PvuII restriction enzyme was employed to genotype the cultured isolates. The results obtained by colonial morphology, PCR, and RD12 methods showed that both isolates were belonging to M. tuberculosis, and Genotyping of the isolates by RFLP technique displayed that two isolates were belonging to different strains of M. tuberculosis.