Noxious stimuli to the ocular surface are encoded by sensory axons of trigeminal ganglion (TG) neurons and conveyed through the ophthalmic branch of the trigeminal nerve (CN V1). We hypothesized that chronic ocular surface pain (COSP) may be associated with microstructural alterations of the trigeminal nerve structures. Our objective was to demonstrate the feasibility of using diffusion tractography to identify and analyze diffusion properties to assess TG microstructure in individuals with and without COSP. Forty COSP patients (27 males and 13 females; mean age: 56.2 ± 11.9 yrs; range: 34-77 yrs) and 17 controls without pain (15 males and 2 females; mean age: 55.4 ± 8.9 yrs, range: 37-66 yrs) were included in the study. Using 3T diffusion MRI (dMRI), we performed tractography to reconstruct TG and CN V1 with a generalized q-sampling imaging (GQI) algorithm. dMRI-based indices such as normalized quantitative anisotropy (NQA), fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were recorded for the right and left TG. TG (n = 45) were successfully reconstructed. Fourteen participants (11 COSP, 3 controls) were excluded because TG could not be clearly visualized. A significant decrease in NQA (p = 0.03), MD (p = 0.04) and RD (p = 0.04) were found in the left TG, but not in the right TG (p = 0.40; p = 0.58; p = 0.64 respectively), in the COSP group as compared to controls. Decrease in these metrics are generally interpreted as indicators of axonal loss and fiber integrity, suggesting the presence of fiber damage in the left TG of patients with COSP.