The fibularis longus muscle, divided into anterior and posterior neuromuscular regions, plays a significant role in eversion strength deficits in individuals with chronic ankle instability (CAI). However, it is unknown whether the muscle fiber conduction velocity (MFCV) of these neuromuscular regions is different between individuals with CAI and those without a history of ankle sprain. This study aimed to compare the MFCV of the anterior and posterior neuromuscular regions of the fibularis longus between individuals with CAI and healthy individuals. A case-control study was conducted. Thirty-five male volunteers were included in the analysis (CAI group: n = 18; No-CAI group: n = 17). High-density surface electromyography (HD-sEMG) was used to record the sEMG amplitude and to calculate the MFCV of the fibularis longus neuromuscular regions (anterior and posterior) during eversion at different contraction intensities. The findings revealed that individuals with CAI exhibited significantly lower MFCV in the posterior region at moderate (30% and 50% maximum voluntary isometric contraction [MVC]) and high (70% and 100% MVC) contraction intensities compared with healthy individuals. Within the CAI group, the posterior neuromuscular region also demonstrated a lower MFCV than the anterior neuromuscular region. These findings indicate that CAI is associated with specific regional electrophysiological changes in the fibularis longus muscle, particularly in the posterior region. Understanding these alterations can inform targeted rehabilitation strategies aimed at improving muscle function and stability in CAI patients. Future research should explore the mechanisms underlying these changes to develop more effective therapeutic interventions.