Background: Human locomotion involves the coordinated activation of a finite set of modules, known as muscle synergy, which represent the motor control strategy of the central nervous system. However, most prior studies have focused on isolated muscle activation, overlooking the modular organization of motor behavior. Therefore, to enhance comprehension of muscle coordination dynamics during multi-joint movements in chronic ankle instability (CAI), exploring muscle synergies during landing in CAI patients is imperative. Methods: A total of 22 patients with unilateral CAI and 22 healthy participants were recruited for this research. We employed a recursive model for second-order differential equations to process electromyographic (EMG) data after filtering preprocessing, generating the muscle activation matrix, which was subsequently inputted into the non-negative matrix factorization model for extraction of the muscle synergy. Muscle synergies were classified utilizing the K-means clustering algorithm and Pearson correlation coefficients. Statistical parameter mapping (SPM) was employed for temporal modular parameter analyses. Results: Four muscle synergies were identified in both the CAI and healthy groups. In Synergy 1, only the gluteus maximus showed significantly higher relative weight in CAI compared to healthy controls (p = 0.0035). Synergy 2 showed significantly higher relative weights for the vastus lateralis in the healthy group compared to CAI (p = 0.018), while in Synergy 4, CAI demonstrated significantly higher relative weights of the vastus lateralis compared to healthy controls (p = 0.030). Furthermore, in Synergy 2, the CAI group exhibited higher weights of the tibialis anterior compared to the healthy group (p = 0.042). Conclusions: The study suggested that patients with CAI exhibit a comparable modular organizational framework to the healthy group. Investigation of amplitude adjustments within the synergy spatial module shed light on the adaptive strategies employed by the tibialis anterior and gluteus maximus muscles to optimize control strategies during landing in patients with CAI. Variances in the muscle-specific weights of the vastus lateralis across movement modules reveal novel biomechanical adaptations in CAI, offering valuable insights for refining rehabilitation protocols.