This study was designed to compare intraoperative fluoroscopic stress testing, static radiographs, and biomechanical criteria for the diagnosis of distal tibiofibular syndesmotic instability associated with external rotation type ankle fractures. Prospective, consecutive series. Academic level 1 trauma center. Thirty-eight skeletally mature patients with unstable unilateral external rotation ankle fractures were prospectively recruited. Before surgery, the treating surgeon detailed the operative treatment plan, including need for syndesmotic fixation. In pronation-external rotation injuries, biomechanical criteria were applied to predict syndesmotic instability. Ankles were examined using intraoperative fluoroscopic external rotation stress tests. The contralateral uninjured limb was used as a control. A 7.2-Nm force was applied for the external rotation stress examination. Stress testing was performed after lateral malleolar fixation and repeated after medial and syndesmotic fixation. The incidence of syndesmotic instability was determined based on radiographic clear space measurements and compared with previously published criteria. Intraoperative fluoroscopy detected unpredicted syndesmotic instability in 37% of ankles. In supination-external rotation (OTA 44B) injuries, unpredicted syndesmosis instability was found in 10 of 30 patients (33%). In pronation-external rotation injuries (OTA 44C), 4 of 7 patients (57%) were associated with syndesmosis disruption not predicted by biomechanical criteria. In bimalleolar fractures, syndesmosis fixation improved stability compared with rigid bimalleolar fixation alone (P < 0.01). Preoperative radiographs and biomechanical criteria are unable to routinely predict the presence or absence of syndesmosis instability. Rigid bimalleolar fixation was frequently not sufficient to stabilize syndesmotic disruption. Intraoperative stress fluoroscopy is a valuable tool for detection of unstable syndesmotic injuries.