Context: Hamstring strain injury (HSI) is the most prevalent injury in football (soccer), and a few intrinsic factors have been associated with higher injury rates. Objective: To describe the prevalence of the main intrinsic risk factors for HSI in professional and under-20 football players. Design: Cross-sectional study. Setting: Physiotherapy laboratory, Federal University of Health Sciences of Porto Alegre (Brazil). Participants: A total of 101 football players (52 professional and 49 under-20 players). Intervention: An evidence-based testing protocol for screening HSI risk factors. Main Outcome Measures: Anamnesis, ultrasonography of the hamstrings, passive straight-leg raise test, Functional Movement Screen, and isokinetic dynamometry were performed. Eleven HSI risk factors for each leg were assessed, besides the player’s age as a systemic risk factor. Reports were delivered to the coaching staff. Results: Professionals had greater prevalence of HSI history compared with under-20 players (40% vs 18%). No between-group differences were found for the other screening tests. Altogether, 30% of players had already sustained at least one HSI; 58% had a history of injuries in adjacent regions; 49% had short biceps femoris fascicles; 66% and 21% had poor passive and active flexibility, respectively; 42% and 29% had deficits in functional movements and core stability, respectively; 7% and 26% presented bilateral imbalance for hamstring concentric and eccentric strength, respectively; 87% and 94% obtained low values for hamstring-to-quadriceps conventional and functional ratios, respectively. Two-thirds of players had 3 to 5 risk factors per leg. None of the players was fully free of HSI risk factors. Conclusion: Most football players present multiple risk factors for sustaining an HSI. Hamstring weakness is the most prevalent risk factor, but the teams should also be aware of deficits in flexibility, core stability, functional movements, and hamstring fascicle length.