Objective: To study genetic bases and morphology of pili in Mycobacterium tuberculosis (M. tuberculosis). Methods: PCR and sequencing were used to investigate two related pili, Mtp and Flp genes in clinical isolates of M. tuberculosis. The primers were designed and PCR program were set for whole genes amplification. PCR products of the two genes from all isolates were sequenced by an applied biosystems apparatus and the results were analysed by online software. In the other hands, harvested cells from fresh cultures of isolates were undergoing specific sample preparation for sectional and negative staining for transmission electron microscopy. Results: Electrophoresis revealed two specific bonds of 361 bp for Mtp and 150 bp for Flp genes and confirmed primer and PCR conditions designing. There were not any mutations in sequencing results of Mtp and Flp in comparison with reference sequence. Transmission electron microscopy examination revealed two distinct types of pili in the isolates as a bundle-forming pilus and rope-like pilus. From total investigated cells, 10% harbored pili in their structure. Conclusions: Two genes of pili in all clinical isolates of M. tuberculosis were conserved and two morphological types of pili were detected. We proposed that by targeting pili proteins by a suitable inhibitor, it could affect the pathogenesis especially in resistant forms.