Objective: To quantitatively assess biochemical alterations in the cartilage of the subtalar and midtarsal joints in chronic lateral ankle instability (CLAI) patients with isolated anterior talofibular ligament (ATFL) injuries and combined calcaneofibular ligament (CFL) injuries using MRI T2 mapping. Materials and Methods: This study was performed according to regulations of the Committee for Human Research at our institution, and written informed consent was obtained from all participants. Forty CLAI patients (26 with isolated ATFL injuries and 14 with combined ATFL and CFL injuries) and 25 healthy subjects were recruited for this study. All participants underwent MRI scans with T2 mapping. Patients were assessed with the American Orthopedic Foot and Ankle Society (AOFAS) rating system. The subtalar and midtarsal joints were segmented into 14 cartilage subregions. The T2 value of each subregion was measured from T2 mapping images. Data were analyzed with ANOVA, the Student’s t test, and Pearson’s correlation coefficient. Results: T2 values of most subregions of the subtalar joint and the calcaneal facet of the calcaneocuboid joint in CLAI patients with combined CFL injuries were higher than those in healthy controls (all p < 0.05). However, there were no significant differences in T2 values in subtalar and midtarsal joints between patients with isolated ATFL injuries and healthy controls (all p > 0.05). Moreover, T2 values of the medial talar subregions of the posterior subtalar joint in patients with combined CFL injuries showed negative correlations with the AOFAS scores (r = -0.687, p = 0.007; r = -0.609, p = 0.021, respectively). Conclusion CLAI with combined CFL injuries can lead to cartilage degeneration in subtalar and calcaneocuboid joints, while an isolated ATFL injury might not have a significant impact on the cartilage in these joints.