Understanding the levator ani complex architecture is of major clinical relevance. The aim of this study was to determine the feasibility of magnetic resonance (MR) fiber tractography with diffusion tensor imaging (DTI) as a tool for the three-dimensional (3D) representation of normal subdivisions of the levator ani. Ten young nulliparous female volunteers underwent DTI at 1.5 T MR imaging. Diffusion-weighted axial sequence of the pelvic floor was performed with additional T2-weighted multiplanar sequences for anatomical reference. Fiber tractography for visualization of each Terminologia Anatomica-listed major levator ani subdivision was performed. Numeric muscular fibers extracted after tractography were judged as accurate when localized within the boundaries of the muscle, and inaccurate when projecting out of the boundaries of the muscle. From the fiber tracking of each subdivision the number of numeric fibers (inaccurate and accurate) and a score (from 3 to 0) of the adequacy of the 3D representation were calculated. All but two volunteers completed the protocol. The mean number of accurate fibers was 17 ± 2 for the pubovisceralis, 14 ± 6 for the puborectalis and 1 ± 1 for the iliococcygeus. The quality of the 3D representation was judged as good (score = 2) for the pubovisceralis and puborectalis, and inaccurate (score = 0) for the iliococcygeus. Our study is the first step to a 3D visualization of the three major levator ani subdivisions, which could help to better understand their in vivo functional anatomy.