BACKGROUND: The prevalence and impact of chronic ankle instability (CAI) in adolescent athletes are unknown. To better develop and justify prevention strategies of lateral ankle sprains and CAI, it is important to understand the origin and associated long-term impact of CAI within populations other than adults. PURPOSE/HYPOTHESIS: The purpose of this study was to determine the prevalence and impact of CAI on ankle function, health-related quality of life (HRQoL), and physical activity in adolescent athletes. The hypothesis was that the presence of CAI will be commonly reported among adolescent athletes and that participants with CAI will have lower self-reported ankle function, HRQoL, and physical activity when compared with participants without CAI. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: A cohort of 1002 healthy (able to fully participate) adolescent athletes (50.4% female; mean age, 15.6 ± 1.6 years) across 8 club sport facilities and high schools completed paper-and-pencil surveys to establish the presence of CAI (Identification of Functional Ankle Instability [IdFAI]) and estimate perceived ankle function (Foot and Ankle Ability Measure [FAAM]-Activities of Daily Living and FAAM-Sport), HRQoL (Pediatric Quality of Life Inventory 4.0 [PedsQL]), and physical activity (Hospital for Special Surgery Pediatric Functional Activity Brief Scale [HSS Pedi-FABS]). RESULTS: The overall prevalence of CAI was 20.0%. Participants with unilateral CAI reported significantly lower ( < .001) ankle function (FAAM-Sport: 87.0 ± 14.8) and HRQoL (total PedsQL: 89.8 ± 9.8) than participants who did not have CAI (FAAM-Sport: 97.7 ± 6.0; total PedsQL: 93.5 ± 9.1). Physical activity was not different between participants with and without CAI. CONCLUSION: The prevalence of CAI was high among adolescent athletes. The presence of CAI negatively affected ankle function and HRQoL in adolescent athletes. Given the high prevalence and negative impact of CAI in an adolescent population, strategies to prevent ankle injuries and maintain physical activity are needed to alleviate future long-term consequences associated with developing CAI. These strategies should be implemented as soon as sport participation begins, as it appears that the origin of CAI may occur before adulthood.