Muscle diffusion tensor imaging is a quantitative magnetic resonance image (MRI) technique, which can provide information about muscular microstructure and integrity. Due to high intermuscular variability, the manual separation of the muscles is essential. Here, we have compared three methods, which allow assessing of diffusion properties of the thigh muscles. In each of the three investigated methods, two independent raters performed the muscle segmentation. We could show, that volume-based tractography proved to be the most reliable and robust method (ICC = 0.923-0.985) and has the advantage to achieve additional information about muscle architecture.