Purpose: Clear links have been made between both lateral ankle sprains and chronic ankle instability (CAI) and the development of ankle post-traumatic osteoarthritis (PTOA). Current visualization techniques such as arthroscopy and standard MRI are ineffective at quantifying early degenerative changes to the subtalar articular cartilage. The most promising approaches for slowing ankle PTOA progression include early conservative interventions to restore appropriate joint biomechanics. Thus, identifying imaging techniques sensitive to early degenerative changes of the subtalar articular cartilage is needed. Therefore, the purpose of this investigation is to determine if CAI patients have higher T1rho relaxation times and relaxation time variability in their subtalar cartilage compared to uninjured controls. T1rho was chosen because evidence indicates it is a marker of proteoglycan density. Methods: Fifteen CAI (age: 21.2±1.8 years, height: 1.7±.8m, mass: 67.0±7.7kg) and fifteen uninjured controls (age: 21.0±2.5 years, height: 1.7±.8m, weight: 69.6±13.2kg) volunteered to participate. CAI inclusion criteria was in accordance with the International Ankle Consortium guidelines. Relative to the controls, CAI participants had a history of multiple lateral ankle sprains (4.0±2.1 vs. 0.0±0.0 sprains), multiple giving way episodes within the past 6 months (6.6±5.1 vs. 0.0±0.0 episodes), higher Identification of Functional Ankle Instability scores (22.9±2.8 vs. 0.1±0.5), and greater limitations in self-reported function based on the Foot and Ankle Ability Measure (85.9±9.7% vs. 100.0±0.0%) and Foot and Ankle Ability Measure Sport (68.4±20.6% vs. 100.0±0.0%). Proteoglycan density was assessed using T1rho MRI with greater T1rho relaxation times interpreted as reduced proteoglycan density. Higher T1rho variability was interpreted as a more diffuse distribution of degenerative changes in the subtalar cartilage. A Siemens Magnetom TIM 3T Prisma scanner and an 8-channel flex coil in conjunction with three-dimensional Fast Low Angle Shot (FLASH) with a spin lock power at 500Hz, five different spin lock durations (40, 30, 20,10, 0 ms) was used to acquire the T1rho MRI. Participants were non-weight bearing for 30 minutes prior to the scan to unload the cartilage. Voxel by voxel T1rho relaxation times were calculated from a five image sequence created with a MATLAB program. Segmentation of the T1rho subtalar cartilage was performed manually using ITK-SNAP software. Three regions of interest: anterior, medial, posterior were identified during segmentation based on talocalcaneal articulations. Mean T1rho relaxation times in each region of interest were compared between CAI patients and healthy controls using independent sample t-tests and an a priori alpha level of 0.05. Results: The CAI and controls groups were similar in terms of their age, height, and weight (p≥0.433). Measures of injury history and self-reported function differed between the groups (p<0.001) as expected. CAI patients had significantly higher T1rho relaxation times in the posterior subtalar articulation (CAI: 65.05±4.76ms, Control: 59.81±5.96ms, p=0.013) but not the anterior (CAI: 66.98±13.21ms, Control: 65.90±11.45ms, p=0.813) or medial articulation (CAI: 69.60±7.70ms, Control: 64.62±7.84ms, p=0.091). CAI patients had significantly higher T1rho relaxation time variability in the medial subtalar articulation (CAI: 32.46±4.58ms, Control: 28.71±5.10ms, p=0.013) but not the anterior (CAI: 35.62±4.87ms, Control: 31.51±8.27ms, p=0.813) or posterior articulation (CAI: 27.33±5.23ms, Control: 23.93±5.74ms, p=0.091). Conclusions: CAI patients demonstrate higher T1rho relaxation times and variability compared to controls in at least one subtalar joint articulation suggesting reduced proteoglycan density in the subtalar cartilage. This finding supports the hypothesis that early compositional changes, potentially indicative of cartilage degeneration, exist in individuals with CAI in subtalar joint. This finding is also consistent with the research indicating compositional changes at the subtalar joint and in on the talar dome in those with CAI. Further research is needed to determine the underlying mechanisms of these deleterious compositional changes and to identify therapeutic interventions capable of slowing the progression of these compositional changes following lateral ankle sprains and CAI.