Thyme (Thymus vulgaris L.) is xerophytes plant that can tolerat against drought and water loss stress easily. Most important secondary metabolites in Thyme are Thymol that have medicinal, antioxidant and antimicrobial properties. No research has carried out so far on the effects of cold stress on the synthesis of Thymol pathway genes and on the physiological and biochemical characteristics. This study was designed to investigate the effect of low temperature stress (4 °C) on the expression Thymol pathway genes such as TPS2 and TPS5 genes using RT-PCR in the time period of 0, 3, 12, 24 and 48 hours after treatment. To evaluate the levels of relative expression of these genes for the synthesis of Thymol (MVA, MEP) t-test was used. TPS2 gene has highest expression within the 3 hours after treatment (3.46 times) compared to the control.