Objective To investigate the morphological characteristics of the os subfibulare (OSF) and evaluate its clinical association with chronic ankle instability (CAI). Methods Imaging data of 130 patients with OSF between January 2015 and August 2025 were retrospectively analyzed, including 82 males and 48 females with a mean age of 30.6 years (range, 10-80 years). Patients were divided into CAI group (n=74) and non-CAI group (n=56). X-ray films were used for screening, while CT images were used to measure the parameters related to the shape and spatial location of the OSF, including the maximum diameter, maximum area, distance from the OSF center to the fibular tip, distance from OSF to the talus, and the angle between the OSF and the fibular long axis. OSFs were classified as regular or irregular. MRI categorized OSF location into three zones based on ligament attachment sites: zone Ⅰ [anterior talofibular ligament (ATFL)], zone Ⅱ (calcaneofibular ligament), and zone Ⅲ (posterior talofibular ligament). Bone interface fluid signal, bone marrow edema, and ATFL injury were recorded. The patients with CAI were stratified and analyzed to compare the differences in the location, shape and spatial localization of the OSF (the maximum diameter of OSF, the distance from the OSF center to the fibular tip, the angle between the OSF and the fibular long axis) and MRI signs between different genders and between different affected sides. ResultsCT measurements showed that, compared with the non-CAI group, the CAI group exhibited differences in spatial localization of the OSF. The distance from the OSF center to the fibular tip was significantly greater in the CAI group (P<0.05), whereas the distance from OSF to the talus, and the angle between the OSF and the fibular long axis showed no significant difference (P>0.05). Regarding morphology and size, the maximum diameter of OSF was significantly larger in the CAI group (P<0.05), while no significant difference was found in maximum area of OSF or morphological type (P>0.05). MRI findings showed that OSFs were predominantly located in zone Ⅰ in both groups, followed by zones Ⅱ and Ⅲ. There was no significant difference in distribution between groups (P>0.05). The incidences of bone marrow edema, bone interface fluid signal, and ATFL injury were significantly higher in the CAI group than in the non-CAI group (P<0.05). Within the CAI group, no significant difference was observed between genders or affected sides in terms of OSF location, morphology, spatial parameters, or MRI findings (P>0.05). Conclusion Patients with CAI showed a larger maximum OSF diameter and a greater distance from the OSF center to the fibular tip, and were more frequently accompanied by MRI findings such as bone marrow edema, bone interface fluid signal, and ATFL injury. These imaging characteristics may help evaluate the relationship between OSF and CAI from an imaging perspective.