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Background: Hamstring strain injuries (HSIs) are prevalent in US National Football League (NFL) players, but there is a paucity of information regarding imaging characteristics, injury severity, and player factors associated with time missed and risk of recurrent injury., Purpose: To describe player, football activity, clinical, and imaging characteristics of NFL players with HSIs, as well as determine player characteristics, clinical examination results, and magnetic resonance imaging (MRI) findings associated with injury occurrence, severity, and missed time., Study Design: Cross-sectional study; Level of evidence, 3., Methods: A retrospective cohort of NFL players with acute HSI (n = 180) during the 2018-2019 season was identified. Injury data were collected prospectively through a league-wide electronic health record system. Three musculoskeletal radiologists graded MRI muscle injury parameters using the British Athletics Muscle Injury Classification (BAMIC) system. Player, football, clinical, and imaging characteristics were correlated with HSI incidence and severity and with missed time from sport., Results: Of the 1098 HSIs identified during the 2018-2019 season, 416 (37.9%) were randomly sampled, and 180 (43.3%) had diagnostic imaging available. Game activity, preseason period, and wide receiver and defensive secondary positions disproportionately contributed to HSI. The biceps femoris was the most commonly injured muscle (n = 132, 73.3%), followed by the semimembranosus (n = 24, 13.3%) and semitendinosus (n = 17, 9.4%) muscles. The most common injury site was the distal third of the biceps femoris and semitendinosus muscles (n = 60, 45.5% and n = 10, 58.8%, respectively) and central part of the semimembranosus muscle (n = 17, 70.8%). Nearly half of the injuries (n = 83, 46.1%) were BAMIC grade 2; 25.6% (n = 46), grade 3; and 17.8% (n = 32), grade 4. MRI showed sciatic nerve abnormality in 30.6% (n = 55) of all HSIs and 81.3% (n = 26) of complete tendon injuries. BAMIC grade correlated with both median days and games missed. Combined biceps femoris and semitendinosus injuries resulted in the highest median days missed (27 days)., Conclusion: Among NFL players with acute HSIs, the most common injury was a moderate-severity injury of the distal biceps femoris. BAMIC grade was associated with missed time., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: M.A.D. has received support for education from Great Lakes Orthopedics, Smith & Nephew, and Arthrex; grant from Arthrex; and hospitality payments from Stryker and Encore Medical. V.K. has received consulting fees from Hyalex Orthopaedics. A.B. has received royalties from Arthrex; consulting fees from Arthrex; and speaking fees from Synthes GmbH. S.A.R. is an associate editor for AJSM and has received consulting fees from Teladoc, DJO, and Novartis Pharmaceuticals Corporation; speaking fees from Terumo BCT and Smith & Nephew; and holds stock or stock options in Ortho RTI. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.