Thymus is one of the most important medicinal plants which belongs to Lamiaceae family. The medicinal properties of genu Thymus include antibacterial, antioxidant, carminative, digestive and anti-inflammatory. Despite the importance of Thymus ecies in terms of distribution and secondary metabolites, a limited information is available on the phylogenetic relationships of some species. Recent advances in molecular studies have provided ample opportunities to answer many questions about species classification, phylogenetic and evolutionary relationships. Hence, this study was performed in two ways (Maximum likelihood and Bayesian) aimed to investigate the phylogenetic relationships and estimation of divergence time of Thymus ecies and Zataria multiflora y using chloroplast (CHL) and single copy orthologs (SCO) information. In this regard, after RNA extraction, the samples were sent to the relevant company to construct the cDNA library and sequencing. After evaluating the quality and assembling reads from sequencing, chloroplast and single copy orthologs sequences were extracted and used for phylogenetic tree construction. Finally,the most reliable topology for reconstructing evolutionary relationships among species was selected by comparing different methodologies and data.Our results indicated that all phylogenetic trees support a close genetic relationship betwee T. daenensis and T. vulgaris (clade1) as sister species and Z. multiflora ecies placed outside of genu Thymus group. Also, the results of divergence times estimation analysis for both genus showed that Zataria has been originated many years earlier than the genu Thymus , so Zataria doesn't fall in to the same group with genu Thymus ( Zataria ~ 17.2 million years ago and Thymus ~ 2 million years ago). Comparison of CHL and SCO data along with tree construction methods showed that the Bayesian phylogenetic tree derived from SCO data, more reliably displays the relationships among species and divergence time estimation. In this tree, the divergence times of Thymus ecies and the genu Zataria were estimated 2 and 17.29 million years ago, respectively. Keywords: Thymu s, Phylogenetic relationships, Divergence time, Chloroplast sequences, single copy orthologous