Due to its accessibility, efficacy, and affordability, traditional medicine (TM) is the main source of health services for many people in the world. Nevertheless, in spite of its benefits, there are still many issues about the principles of TM which demand further declaration. One of the essential principles of Iranian traditional medicine (ITM) is temperament (mizaj), which efficiently applied in diagnosis and therapy of illnesses. In this study we aimed to explore the association of GSTM1/T1, and SOD1 50 bp Ins/Del polymorphisms with combined groups of temperament. The study was conducted in 217 healthy males from Fars province, southern Iran. The self-reported mizaj questionnaire was applied to identify the participants' temperament. Then individuals with temperate, warm/moist, and warm/dry temperament were entered in the study. To determine the genotype of GSTM1, GSTT1, and SOD1, the polymerase chain reaction (PCR)-based method was performed. As the results of χ2 analysis showed, the frequency of GSTT1, GSTM1, and SOD1 polymorphisms in temperate group was not significantly differ from that in each of warm/moist and warm/dry groups. Further research with larger samples are suggested to clarify the association between temperament and biomolecular features.