Background and Objectives: Increasing the concentration of carbon dioxide (CO2) leads to reduced comfort and learning disruption. Therefore, the present study was conducted to determine the concentration of CO2 in classrooms of Babol and Gonabad cities and its relationship with classroom ventilation and temperature. Materials and Methods: This descriptive study was randomly carried out in spring of 2018 in 6 high schools and 28 classrooms of Babol in the north of Iran and 28 educational classes in Gonabad University of Medical Sciences in the east of Iran. A CO2 meter was used to measure CO2. In order to determine the physical parameters affecting CO2 concentration, information of a checklist was used. Data were analyzed by statistical tests such as Chi-square, ANOVA and t-test. Results: In the present study, there was a significant correlation between CO2 and ventilation type (p = 0. 021) and heating system in Babol schools (p = 0. 025). There was a significant relationship between CO2 concentration in school and university classes (p=0. 001) in Babol and Gonabad. Conclusion: The present study showed that CO2 concentration was high in most classes and it was related to the type of ventilation and heating and cooling systems.