The relationship between asymmetrical occlusion and surface electromyographic activity (sEMG) in people with different chewing preferences is not clear. In this study, the 5 s sEMG changes in the masseter muscle (MM), sternocleidomastoid (SCM), lateral (LGA), and medial (MGA) gastrocnemius muscles were recorded in controls, and subjects with chewing side preference (CSP) during clench with bilateral (BCR), left (LCR), and right (RCR) posterior teeth placement of cotton rolls. The images of the middle 3 s were selected and expressed as the root mean square (unit: μV/s). The EMG waves of bilateral muscles were compared by computing the percentage overlapping coefficient (POC). Only the POCMM of the CSP showed gender differences at BCR and RCR. Between the control group and the CSP group, there were significant differences in the POCMM and the POCLGA at BCR. In addition, there was a significant difference in POCMM and POCSCM between the two populations in different occlusal positions. The change in the POCSCM correlated with the change in the POCMM (r = 0.415,p= 0.018). The experiment-induced asymmetrical occlusion showed that the altered symmetry of the MM correlated with the altered symmetry of the SCM. Long-term asymmetrical occlusion (i.e., CSP) not only affects MM but also has potential effects on other superficial muscles (e.g., LGA).