Materials and Objectives: Mycobacterium tuberculosis is an important bacterial pathogen which causes ‘‘global health emergency’’ according to the World Health Organization. DNA sequencing has greatly enhanced the bacterial identification. In this way molecular methods using sequences of one or several appropriate genes are gaining importance because they yield quick and accurate results. Sequence-based genotyping methods such as Multilocus sequence analysis (MLSA) need appropriate genes for an acceptable result. There is no standard target for phylogenetic study of M. tuberculosis yet. In this study, we introduce a good target for sequence analysis of clinical isolates of M. tuberculosis. Materials and Methods: In order to assess evolutionary and phylogenetic relations of Mycobacterium tuberculosis isolates, several gene targets were evaluated. In this study, appropriate fragments of 5 highly variable genes (rpsL, mprA, lipR, katG, and fgd1 genes) were sequenced. The sequence data were analyzed with neighbor-joining method using mega and Geneious software. Results: The phylogenetic trees analyses revealed that the discriminatory power of lipR is much stronger than that observed in the other genes. lipR could distinguish between more clinical isolates. Six distinct phylogenetic groups were distinguished by lipR. Therefore, lipR can serve as a promising target for sequence analysis of M. tuberculosis. Conclution: DNA sequencing analysis has allowed the detection of thousands of single nucleotide polymorphisms (SNPs) in clinical isolates of each organism. During recent years these genetic variations have been widely used for strain identification and epidemiological studies. The selection of a proper gene with high discriminatory power plays a critical role in such phylogenetic studies. The results showed that the discriminatory power of lipR is much higher than that observed in the other genes. LipR can strongly distinguish between isolates and the resulted trees showed a good power of discrimination. Taken together, we propose lipR gene for phylogenetic study of M. tuberculosis strains as this gene is highly variable and at the same time is present in all M. tuberculosis species.