© 2021, Fondazione Società Italiana di Neurologia.Objective: The aims of this study were (a) to examine the surface electromyography (sEMG) amplitude values of the lower limb muscles during stair climbing both between different functional levels of Duchenne muscular dystrophy (DMD), in comparison with healthy children, and (b) to investigate the relationships between sEMG amplitudes and physical performance. Methods: sEMG amplitudes of the lower limbs of twenty-one children with DMD between levels I and III according to the Brooke Lower Extremity Functional Classification Scale and eleven healthy peers were evaluated by using sEMG during stair climbing task. Physical performance was evaluated by 6-min walk test and ascending 4-step timed performance test. Results: The lower limb sEMG amplitude values of children with DMD were statistically higher than healthy children (p < 0.001). sEMG amplitudes of the right (p = 0.01) and left (p = 0.003) biceps femoris, the right (p < 0.001) and left (p = 0.001) gastrocnemius medialis, and the right vastus lateralis (p = 0.02) muscles were higher in children with levels 2–3 than those in level 1. Moderate-to-strong relations were found between the gastrocnemius medialis and biceps femoris sEMG amplitudes and physical performance assessments (p < 0.05). Conclusion: Increased sEMG amplitude values in the lower limbs during stair climbing task are thought to be caused by the effort to compensate for progressive muscle weakness and are associated with lower physical performance in children with DMD. Further, sEMG amplitude values are determined to increase as the functional level deteriorates. Clinical trial registration number and URL: NCT04287582 (https://clinicaltrials.gov/ct2/show/NCT04287582?term=merve+bora&draw=2&rank=1).