BACKGROUND: Lower limb amputation is a major public health issue globally, and its prevalence is increasing significantly around the world. Previous studies on lower limb amputees showed analogous complexity implemented by the neurological system which does not depend on the level of amputation. RESEARCH QUESTION: What are the differences in muscle synergies between healthy subjects (HS) and transfemoral amputees (TFA) during self-selected normal transient-state walking speed? METHODS: thirteen male HS and eleven male TFA participated in this study. Surface electromyography (sEMG) data were collected from HS dominant leg and TFA intact limb. Concatenated non-negative matrix factorization (CNMF) was used to extract muscle synergy components synergy vectors (S) and activation coefficient profiles (C). Correlation between a pair of synergy vectors from HS and TFA was analyzed by means of the coefficient of determination (R2). Statistical parametric mapping (SPM) was used to compare the temporal components of the muscle synergies between groups. RESULTS: the highest correlation was perceived in synergy 2 (S2) and 3 (S3) and the lowest in synergy 1 (S1) and 4 (S4) between HS and TFA. Statistically significant differences were observed in all of the activation coefficients, particularly during the stance phase. Significant lag in the activation coefficient of S2 (due mainly to activated plantarflexors) resulted in a statistically larger portion of the gait cycle (GC) in stance phase in TFA. SIGNIFICANCE: Understanding the activation patterns of lower limb amputees' muscles that control their intact leg (IL) and prosthetic leg (PL) joints could lead to greater knowledge of neuromuscular compensation strategies in amputees. Studying the low-dimensional muscle synergy patterns in the lower limbs can further this understanding. The findings in this study could contribute to improving gait rehabilitation of lower limb amputees and development of the new generation of prostheses.