: Insufficient light intensity in the classroom might be related to asthenopia in students, and consequently have a detrimental impact on learning performance. The aim of this study was to measure light intensity in classrooms, and to investigate the factors associated with asthenopia among 507 preclinical medical students. Light intensity was measured at all seats in classrooms, and compared to the standard issued by Department of Labour Protection and Welfare, Thailand. Asthenopia and associated factors were also investigated. The average light intensity in the 1st year classroom passed the standard, while lighting in the 2nd and 3rd year classrooms failed to reach the standard. The remarkable findings were that the students who sit at the seat with lower spotlight intensity had a significantly higher prevalence of asthenopia (OR=0.993, 95%CI=0.989-0.998). Female had higher prevalence of asthenopia than male (OR=3.571, 95%CI=1.602,7.958). The students who had myopia had higher prevalence of asthenopia than the students who had normal sight (OR=2.694, 95%CI=1.205,6.022). Therefore, the results can be used to conclude that insufficient light intensity in classrooms had an effect on asthenopia among preclinical medical students.