Dysregulation and fibrosis of the extracellular matrix (ECM) in skeletal muscle is a consequence of injury. Current ECM assessment necessitates muscle biopsies to evaluate alterations to the muscle ECM, which is often not practical in humans. The goal of this study was to evaluate the potential of a magnetic resonance imaging sequence that quantifies T1ρ relaxation time to predict ECM collagen composition and organization. T1ρ imaging was performed and muscle biopsies obtained from the involved and non‐involved vastus lateralis muscle on 27 subjects who had an anterior cruciate ligament (ACL) tear. T1ρ times were quantified via monoexponential decay curve fitted to a series of T1ρ‐weighted images. Several ECM indices, including collagen content and organization, were obtained using immunohistochemistry and histochemistry in addition to hydroxyproline. Model selection with multiple linear regression was used to evaluate the relationships between T1ρ times and ECM composition. Additionally, the ACL‐deficient and healthy limb were compared to determine sensitivity of T1ρ to detect early adaptations in the muscle ECM following injury. We show that T1ρ relaxation time was strongly associated with collagen unfolding (t = 4.093, P = 0.0007) in the ACL‐deficient limb, and collagen 1 abundance in the healthy limb (t = 2.75, P = 0.014). In addition, we show that T1ρ relaxation time is significantly longer in the injured limb, coinciding with significant differences in several indices of collagen content and remodelling in the ACL‐deficient limb. These results support the use of T1ρ to evaluate ECM composition in skeletal muscle in a non‐invasive manner.