Diffusion tensor imaging (DTI) has been used to indicate the direction of nerve and muscle fibers by using the characteristics that water molecules preferentially diffuse along the fibrous structure. However, DTI fiber tractography for multipennate muscles, such as the masseter muscle, is challenging due to a lack of data regarding the imaging parameters. This study aimed to determine the optimal DTI parameters for masseter muscle fiber tractography. A 27-year-old healthy man voluntarily underwent DTI and T1-weighted magnetic resonance imaging of the right masseter muscle. Four imaging parameter settings were created by combining the following parameters that particularly affect the signal-to-noise ratio: b-value, number of excitations (NEX), and number of motion probing gradient (MPG) directions. DTI fiber tractography was performed using specific software for each parameter setting. The length and orientation of the muscle fibers in each layer were calculated. As a result, the masseter muscle fibers of each layer were identified on DTI. Although the detected fiber length was affected significantly by the imaging parameters, the fiber orientation was insignificantly affected. The appropriate combination of the b-value, NEX, and the number of MPG directions for masseter muscle fiber tractography could be determined based on previously reported anatomical data of the masseter muscle fibers. DTI may enable the non-invasive evaluation of masseter muscle fiber length and orientation. Elucidation of the details of masseter muscle fiber orientation is useful in evaluating stomatognathic biomechanics and muscle disorders.