Background: Prior research has shown differences in physiologic and behavioral measurements of adolescent boys and girls. This study sought to determine if similar gender-specific differences exist between participants of the Project Healthy Schools (PHS) school-based wellness program, both at baseline and post-PHS intervention. Methods: Demographics, physiological measures and health behaviors were compared between 6 th grade girls and boys enrolled in PHS between 2004-2017 at baseline and post-PHS intervention. Results: Health behavior data were collected in 19,995 students (46.1% girls); physiological measures were collected in 2,922 students (52.7% girls). Boys within the physiological sample were older than girls (11.62 v. 11.55, p<0.001). At baseline, girls had lower HDL cholesterol and higher resting and recovery heart rates, but reported greater consumption of fruits and vegetables and less sugary beverages and fried meats than boys. Boys reported more vigorous exercise but greater hours of television and video game screen time than girls. Post-intervention, girls showed greater improvement in resting heart rate, fruit consumption and video game time. Conclusion: Girls had better health behaviors but poorer physiological markers at baseline. Lower rates of physical activity in girls at baseline highlight a potential area of focus for the PHS intervention in this population. Future exploration of gender-specific targeted strategies to improve health behaviors is warranted.