Objective To explore the differences in muscle synergies of healthy individuals, intact limbs and residual limbs of above-knee amputees under different gaits, provide references for electromyographic control of the prosthetics. Methods Surface electromyography (sEMG) signals of 7 muscles (4 thigh muscles, 3 calf muscles) were collected from 7 healthy individuals and 3 lower-limb amputees during walking, ascending/descending slopes, and ascending/descending stairs. Muscle synergy weights (W) and temporal activation coefficients (H) were extracted using concatenated non-negative matrix factorization (CNMF). The weight matrices were compared using the coefficient of determination R2, and the activation coefficients were analyzed using statistical parametric mapping (SPM). Furthermore, peak activation and activation integral were used for hierarchical clustering analysis. Results The comparison of W matrices indicated a similar set of muscle synergies between the healthy group and the intact limb of amputees, whereas the residual limb showed greater variability in synergies. The analysis of H showed statistically significant differences in muscle activation throughout the gait cycle for all amputees. Clustering of time-domain features revealed that amputees exhibited higher activation integral and peak values in their intact limb during ascending slopes. Conclusions This study provides an in-depth investigation of the neuromuscular compensation strategies in lower-limb amputees under different gaits, offering theoretical insights for gait rehabilitation and assisting in the development of EMG-controlled prosthetics.