Thymus is one of the most important genera of the Lamiaceae family. This work was performed to assess inter and intra species variation, which is an indispensable prerequisite for the selection and the exploitation of the germplasm, using yield, secondary metabolites, and ploidy level criteria. Nineteen Iranian populations belonging to 11 Thymus species which includes T. vulgaris were used in this study. The results of cytological observations on the 19 populations revealed the three root‐tip chromosome numbers of 2n=2x=30, 2n=4x=56 or 60 (diploid and tetraploid). This study also presents the results of a two‐year field experiment that evaluates the agronomic and morphology of the 19 populations of Thymus spp. Cluster analysis grouped the populations into six groups and explained the relationships among ploidy levels, morphological traits, and essential oils (EOs). In general, diploid species belonged to the thymol chemotype, whilst carvacrol chemotype consistently dependent on the gene‐dosage effect. Thymus migricus, T. daenensis‐2, T. serpyllum, and T. trautvetteri populations with diverse thymol background were the best selection as the parents to improve thymol in a breeding program. Moreover, dry and fresh weight criteria can be used to improve EO content in thyme. Achieving this goal would be expected by crossing T. migricus and T. daenensis‐2. Finally, providing relevant information on the ploidy level of Thymus species, with emphasis on morphology and EO components variations, may be recommended for the selection of populations or species to improve bioactive components as well as morphological traits in future breeding programs.