Objective: Identify key flexibility and point-of-care musculoskeletal ultrasound (POCUS) measures for prognosticating return-to-play (RTP) following a first hamstring strain injury (HSI) and informing the clinical decision–making process. Design: Exploratory prospective cohort study. Setting: Sport medicine and rehabilitation clinic of a Canadian university. Participants: One hundred and sixty-seven elite Canadian university football athletes followed over 5 seasons. Interventions: Clinical and POCUS measures collected within 7 days after HSI and preseason clinical measures. Main Outcome Measures: Active knee extension (AKE) and Straight Leg Raise (SLR) to quantify hamstring flexibility, POCUS-related outcomes to characterize tissue alteration, and RTP until full sport resumption were documented (categorized as Early [1-40 days] or Late [>40 days] RTP). Results: A total of 19 and 14 athletes were included in the Early RTP (mean RTP = 28.84 ± 8.62 days) and Late RTP groups (mean 51.93 ± 10.54 days), respectively, after having been diagnosed with a first HSI. For the clinical results, height and a greater flexibility asymmetry measure with the AKE or SLR when compared with both ipsilateral preseason and acute contralateral values significantly increases the chance of facing a long delay before returning to play (ie, RTP). For the POCUS-related results, the Peetrons severity score, extent of the longitudinal fibrillary alteration, and novel score lead to similar results. Conclusions: Early hamstring flexibility asymmetry following acute HSI, particularly the AKE, along with some POCUS-related measures are valuable in prognosticating late RTP following among Canadian university football athletes.