To characterize the levator ani muscle (LAM) injury after first vaginal delivery and investigate the clinical application of diffusion tensor imaging (DTI) and fiber tractography in evaluating the LAM. Fifty-eight primiparous women at 6weeks after vaginal delivery and 27 nulliparous women as controls underwent T2-weighted sequence and DTI sequence of the pelvic floor. A LAM scoring system was used to characterize the morphological changes. Fiber tractography of each major subdivision of LAM was performed, followed by assessment of the quality of fiber tracking. Fractional anisotropy (FA), apparent diffusion coefficient (ADC), fiber volume, and length were calculated. Puborectalis and iliococcygeus injuries were observed in 30/58 (51.7%) and 10/58 (17.2%) primiparae, respectively. No LAM defects were identified in the control group. For the puborectalis, the FA values were lower (P=0.010) and ADC values were higher (P=0.024) in the primiparous group than in the control group. For the iliococcygeus, the fiber volume values were lower in the primiparous group than in the control group (P=0.004). Vaginal delivery can result in LAM injury at the puborectalis. DTI parameters can assist in the quantitative diagnosis of the LAM injury.