OBJECTIVES: To prospectively explore the clinical application of diffusion tensor imaging (DTI) and fibre tractography in evaluating the pelvic floor. METHODS: Ten patients with pelvic organ prolapse, ten with pelvic floor symptoms and ten asymptomatic women were included. A two-dimensional (2D) spin-echo (SE) echo-planar imaging (EPI) sequence of the pelvic floor was acquired. Offline fibre tractography and morphological analysis of pelvic magnetic resonance imaging (MRI) were performed. Inter-rater agreement for quality assessment of fibre tracking results was evaluated using weighted kappa (κ). From agreed tracking results, eigen values (λ1, λ2, λ3), mean diffusivity (MD) and fractional anisotropy (FA) were calculated. MD and FA values were compared using ANOVA. Inter-rater reliability of DTI parameters was interpreted using the intra-class correlation coefficient (ICC). RESULTS: Substantial inter-rater agreement was found (κ = 0.71 [95% CI 0.63-0.78]). Four anatomical structures were reliably identified. Substantial inter-rater agreement was found for MD and FA (ICC 0.60-0.91). No significant differences between groups were observed for anal sphincter, perineal body and puboperineal muscle. A significant difference in FA was found for internal obturator muscle between the prolapse group and the asymptomatic group (0.27 ± 0.05 vs 0.22 ± 0.03; P = 0.015). CONCLUSION: DTI with fibre tractography permits identification of part of the clinically relevant pelvic structures. Overall, no significant differences in DTI parameters were found between groups. KEY POINTS: • Diffusion tensor MRI offers new insights into female pelvic floor problems. • DTI allows 3D visualisation and quantification of female pelvic floor anatomy. • DTI parameters from pelvic floor structures can be reliably determined. • No significant differences in DTI parameters between groups with/without prolapse.