Thymus is an important genus of the Lamiaceae family that has several applications in the food, health, cosmetics and especially pharmaceutical industries. The study of diversity (including morphological, phytochemical and cytogenetical) in the next breeding steps including selection, is important. In addition, the study of the phytochemical characteristics of the plants with medicinal properties and the selection of superior population is also an important step in the process of breeding of this plant. One of the strategies to increase the secondary metabolites of the plant is the use of biotic and abiotic factors that can affect the metabolic pathways of secondary metabolites and increase their production. To evaluate the pheno-morphological traits during the two consecutive years, 19 populations were planted at Randomized Complete Block Design with three replications at Research Farm of Isfahan University of Technology. The result showed a wide range of variation among different population. Additionally, 11 Thymus populations were used to assess the effect of inbreeding on fresh weight, dry weight and essential oil content. In self and outcross pollination derived populations, T. pubescens and T. trautvetteri possessed the highest essential oil content, respectively. Inbreeding increased the essential oil content in T. fallax , T. pubescens , and T. fedtschenkoi2 populations. All studied Thymus population were examined for ploidy level and number of chromosomes. Two ploidy levels (diploid and tetraploid) and three chromosome numbers (30, 56, 60) were recognized. Identifying superior populations with high amount of thymol and carvacrol and same ploidy level as well as genetically distinct ones allows for the identification of cross hybridization containing heterosis. Based on obtained result, can be expected superior progeny production in terms of thymol content from hybridization among T. kotschyanus2 and T. kotschyanus3 . According to the results, the evaluated populations had a high diversity in terms of all phytochemical traits including total phenolic and flavonoid content, antioxidant activity as well as essential oil composition. Gas chromatography–mass spectrometry analysis revealed the presence of 38 compounds in which the major constituents including thymol, carvacrol, geraniol, and p-cymene. The highest amount of thymol was observed in T. migricus under field condition. Furthermore, the essential oils composition of four Thymus species were evaluated under five light spectra (namely, red, blue, red-blue, white, and greenhouse condition). The highest essential oil yield was observed under red light in T. migricus, while the lowest was belonged to T. carmanicus under greenhouse conditions. Light quality also led to difference in essential oil constituents. The highest thymol was found in T. migricus exposed to blue light LED, while the least was observed in T. kotschyanus grown under red-blue light. The LED treatments did not induce any significant effect on carvacrol of Thymus species in comparison to the greenhouse condition. In the present study, the identification of TPS2 is performed for the first time in eight important Iranian Thymus species rich in thymol and carvacrol. The cDNA of the studied species possessed an open reading frame ranging from 1788 to 1794 bp, presenting all the conserved motifs characteristics of monoterpene synthases. Overall, phylogenetic analysis according to whole sequence of Ttps2 gene can help providing insights in respect to its evolutionary process. Keywords: Thymus , Genetic Diversity, phytochemical, Cytogenetic, Light-Emitting Diodes, ?- Terpinene synthase gene.