Purpose To determine if eccentric knee flexor strength and biceps femoris long head (BFlh) fascicle length were associated with prospective hamstring strain injury (HSI) in professional Australian Football players, and if more frequent assessments of these variables altered the association with injury risk. Methods Across two competitive seasons, 311 Australian Football players (455 player seasons) had their eccentric knee flexor strength during the Nordic hamstring exercise and BFlh architecture assessed at the start and end of preseason and in the middle of the competitive season. Player age and injury history were also collected in preseason. Prospective HSIs were recorded by team medical staff. Results Seventy-four player seasons (16%) sustained an index HSI. Shorter BFlh fascicles (<10.42 cm) increased HSI risk when assessed at multiple time points only (relative risk [RR], 1.9; 95% confidence interval [CI], 1.2–3.0). Neither absolute (N) nor relative (N·kg−1) eccentric knee flexor strength was associated with HSI risk, regardless of measurement frequency (RR range, 1.0–1.1); however, between-limb imbalance (>9%), when measured at multiple time points, was (RR, 1.8; 95% CI, 1.1–3.1). Prior HSI had the strongest univariable association with prospective HSI (RR, 2.9; 95% CI, 1.9–4.3). Multivariable logistic regression models identified a combination of prior HSI, BFlh architectural variables and between-limb imbalance in eccentric knee flexor strength as optimal input variables; however, their predictive performance did not improve with increased measurement frequency (area under the curve, 0.681–0.726). Conclusions More frequent measures of eccentric knee flexor strength and BFlh architecture across a season did not improve the ability to identify which players would sustain an HSI.