Objective Medial tibial stress syndrome (MTSS) is a common lower extremity overuse injury often causing long-term reduction of sports participation. This study aimed to investigate the incidence and risk factors of MTSS in first-year Dutch Physical Education Teacher Education (PETE) students. Methods This prospective study consisted of physical measures at baseline (height, weight, fat percentage, 3000 m run test, navicular drop test, hip internal and external range of motion, hip adduction and adduction strength, single leg squat and shin palpation), an intake questionnaire at baseline (age, sport participation, presence of MTSS, MTSS history, insole use and use of supportive shoes) and an MTSS registration procedure during the academic year of 2016-2017 (10months) using a validated questionnaire. In total 221 first-year PETE students were included, of whom 170 (77%) were male and 51 (23%) female. The evaluation of risk factors was conducted with univariable and multivariable logistic generalised estimating equation analyses. Results In total 55 (25%) subjects, 35 (21%) men and 20 (39%) women, developed MTSS during the follow-up period. The associated risk factors were female sex (OR=3.14, 95% CI 1.39 to 7.11), above-average age (OR=0.31, 95% CI 0.13 to 0.76), above-average body mass index (OR=2.29, 95% CI 1.02 to 5.16) and history of MTSS (OR=5.03, 95% CI 1.90 to 13.30). Conclusion The incidence of MTSS is high in PETE students. Several risk factors were identified. These results demonstrate the need for prevention and may provide direction to preventive intervention design.