The aim of the study was to define activation characteristics of knee muscles of the kicking leg during the in-step kick and interpret these in relation to isokinetic strength parameters and ball velocity in professional (N = 14) compared to amateur (N = 17) soccer players. Electromyographic (EMG) recordings were taken from the rectus femoris (RF), vastus lateralis (VL), vastus medialis (VM), long head of biceps femoris (BF) and medial gastrocnemius (GAS) as players performed three successful maximal in-step kicks using a 0 degrees approach angle with two steps to a stationary ball towards a target. Kicks were also recorded with high speed video and ball velocity was measured by a radar gun. Afterward the bilateral concentric isokinetic strength of the knee extensors and flexors was recorded. Results suggest that during the swing phase, BF activation occurred significantly earlier, RF showed reduced activation and an earlier and greater muscle activity occurred in VL and VM in professionals compared to amateur players. During knee extension, VL and VM showed a markedly greater magnitude of muscle activity in professionals; during the follow through, GAS showed reduced activity in professionals. Relationships between muscle strength and performance (ball speed) were found for amateurs but not for professionals (r = 0.57 to r = 0.72, p < 0.05). The findings of this study indicate that the superior performance of professional players compared to amateurs appears not to be due to muscle strength factors but rather to subtle differences in technique, as indicated by EMG data, throughout both the build up and execution of the kick.