Objective The role of the syndesmotic ankle ligaments as extrinsic stabilizers of the distal tibiofibular joint (DTFJ) has been studied extensively in patients with high ankle sprains (HAS). However, research concerning the fibular incisura as intrinsic stabilizer of the DTFJ has been obscured by a two-dimensional assessment of a three-dimensional structure. Therefore, we aimed to compare the morphometry of the incisura fibularis between patients with HAS and a control group using three-dimensional radiographic techniques. Materials and methods Fifteen patients with a mean age of 44 years (SD = 15.2) diagnosed with an unstable HAS and twenty-five control subjects with a mean age of 47.4 years (SD = 6.5) were analyzed in this retrospective comparative study. The obtained CT images were converted to three-dimensional models, and the following radiographic parameters of the incisura fibularis were determined using three-dimensional measurements: incisura width, incisura depth, incisura height, incisura angle, incisura width-depth ratio, and incisura-tibia ratio. Results The mean incisura depth (M = 4.7 mm, SD = 1.1 mm), incisura height (M = 36.1 mm, SD = 5.3 mm), and incisura angle of the control group (M = 137.2°, SD = 7.9°) differed significantly from patients with a HAS (resp., M = 3.8 mm, SD = 1.1 mm; M = 31.9 mm, SD = 3.2 mm; M = 143.2°, SD = 8.3°) ( P <  0.05). The incisura width, incisura width-depth ratio, and incisura-tibia ratio demonstrated no significant difference ( P >  0.05). Conclusion Our three-dimensional comparative analysis has detected a shallower and shorter fibular incisura in patients with HAS. This distinct morphology could have repercussion on the intrinsic or osseous stability of the DTFJ. Future prospective radiographic assessment could determine to what extend the fibular incisura morphology contributes to syndesmotic ankle injuries caused by high ankle sprains.