This cross-sectional study was carried out to explore the potential risk factors of physical activity-related injuries (PARI) among middle-school students of different genders. Selected by the random cluster sampling method, students aged from 12 to 16 years old in grades 7–8 from six middle schools in Shantou, southern China, were recruited for this investigation in November 2017. Information about socio-demographics, physical activity (PA) exposure time, individual exercise behaviors, risk-taking behaviors, and PARI experiences in the past 12 months was collected. A multivariate logistic regression model was used to explore the risk factors of PARI. A total of 1270 students completed valid questionnaires, with an overall injury incidence of 33.6% (boys: 42.0%; girls: 25.0%), an injury risk of 0.68 injuries/student/year, and an injury rate of 1.43 injuries per 1000 PA exposure hours. For boys, living in a school dormitory, participating in sports teams, exercising on a wet floor, rebellious behavior, and having longer PA exposure time were the risk factors of PARI. For girls, those who were sports team members, whose parents were divorced or separated, and those with longer PA exposure time were more vulnerable to suffer from PARI. In conclusion, PARI was a health problem among middle school students in southern China. Boys and girls differed in PARI occurrence and were affected by different risk factors, which provides a basis for targeted gender-specific intervention programs to reduce the occurrence of PARI among middle-school students.