Background: To investigate nerve fibre damage of the visual pathway in patients with primary open-angle glaucoma using tract-based spatial statistical analysis of diffusion tensor imaging and correlate these measures with the clinical severity of glaucoma. Design: Cross-sectional study. Participants: Twenty-five individuals with primary open-angle glaucoma and 24 healthy controls were recruited. Methods: All subjects underwent detailed ophthalmological examinations, including the cup-to-disc ratio, retinal nerve fibre layer thickness and visual fields test. Diffusion tensor imaging of the visual pathway was performed using a 3.0-T magnetic resonance scanner. Main Outcome Measures: Diffusivity changes of the nerve fibres in the visual pathway were calculated through tract-based spatial statistical analysis. The mean diffusivity and fractional anisotropy were assessed and compared with ophthalmological measurements. Results: Compared with controls, bilateral optic tracts and optic radiations in primary open-angle glaucoma patients showed significantly decreased fractional anisotropy and increased mean diffusivity (P < 0.05). In the glaucoma group, the fractional anisotropy of the optic tracts and optic radiations varied consistently with the cup-to-disc ratio, retinal nerve fibre layer thickness and visual function analysis, respectively (P < 0.05). The mean diffusivity of the optic tracts correlated with these ophthalmological measurements (P < 0.05). However, no significant correlation was observed between the mean diffusivity of the optic radiations and the ophthalmological measurements (P > 0.05). Conclusions: The optic tracts and optic radiations of primary open-angle glaucoma patients demonstrated radiological evidence of neurodegeneration. This varied with damage to the optic disc and with the loss of visual function. Tract-based spatial statistical analysis of diffusion tensor imaging is an objective and effective tool for detecting the loss of cortical nerve fibres in primary open-angle glaucoma.