Objective: To explore the feasibility of morphological and functional evaluation of the lateral pterygoid muscle (LPM) by diffusion tensor imaging (DTI) in vivo. Methods: 30 healthy volunteers underwent DTI with the jaw in the rest position, opening and clenching. Diffusion parameters of the superior head of the LPM (SHLP) and the inferior head of the LPM (IHLP) at different jaw positions were calculated. Results: When the jaw was in the rest position, lambda(3) of the SHLP was significantly lower than that of the IHLP; fractional anisotropy (FA) value of the SHLP was significant higher than that of the IHLP. There was no significant difference in lambda(1), lambda(2) and apparent diffusion coefficient (ADC) value. During jaw opening, therewas significant increase of all three eigenvalues and ADC value, and significant decrease of FA value both at the SHLP and IHLP. Clenching caused a significant increase in the ADC and all three eigenvalues, and caused a significant decrease of FA at the SHLP. However, at the IHLP, the variations of all diffusion parameters by clenching in the intercuspal position showed no significance when compared with those at rest. Conclusion: The morphological and functional changes of LPM fibres caused by jaw movements could be sensitively detected by DTI which may serve as a new and non-invasive method for simultaneously investigating the functional and morphological features of the LPM during jaw movement. Advances in knowledge: A new application of DTI is proposed for the morphological and functional evaluation of the LPMs. The results show that the significant change of three eigenvalues indicates the activity of the LPM in a specific jaw movement, a finding that shows the potential value of DTI serving as a new and non-invasive method for investigation of the LPM.