Introduction: The perception of stress by students affects their academic performance significantly. Continuous exposure to stress for a long time can cause both physical illnesses (including heart disease), and mental illnesses (e.g., anxiety disorders). Furthermore, stress increases the risk of suicide in students which is an important cause of adolescent mortality. Perception of stressors depends upon the biological, social, and psychological factors of students. Moreover, biological differences between male and female students attributed to the significant difference in experiencing stress among them. Aim: To study the gender difference and stress levels among adolescent school students. Materials and Methods: A cross-sectional study was conducted in Government boys higher secondary school and Government girls higher secondary school Mauganj, a town of Madhya Pradesh, India, between August 2016 to September 2016. The state board of examinations and the Hindi medium of instruction were followed in the school. Total 40 study subjects, 24 males and 16 females were recruited for study after obtaining written informed consent from participants by simple random sampling method. The self-administered student stress scale was used to measure participants’ levels of stress and socio-demographic information, including age, sex, education, was recorded in semistructured data entry proforma. The data were summarised using means with standard deviations and frequencies with percentages. Comparative analyses between the genders were conducted using the Student’s t-test (for continuous variables). The p-value ≤0.05 was considered level of significance. Results: Total of 40 study participants included, mean age 16.2±2.2 years. A total of 24 were male students and 16 were female students, amongst included subjects. Majority of student’s family practiced Hindu religion. Total stress score was 84.3±7.5 for male and 96.2±5.5 for female students (p=0.001). Conclusion: Significant amounts of stress were felt by students in high school. Male and female students perceived similar levels of stress in their academic and vocational domains, but female students felt significantly more stress in their financial, emotional, familial, and social domain. Student total stress levels were significantly higher for female students than male students.