To compare histological thickness of the retinal nerve fibre layer in the primate with retardation measurements obtained in vivo using the Mark II Nerve Fiber Analyzer (NFA, Laser Diagnostic Technologies, San Diego, USA). Scanning laser polarimetry was performed on both eyes of a healthy anaesthetised adult primate (Macaca mulatta). The retinal nerve fibre layer thickness was measured in the eye with the best polarimetry image. A nerve fibre layer thickness map was scaled and aligned to a retardation map to permit correlation of retardation and thickness measurements. Retinal nerve fibre layer thickness measurements could be satisfactorily aligned with corresponding retardation values at 216 locations. The overall correlation coefficient for nerve fibre layer thickness and retardation was r = 0.70 (n = 216, p < 0.001). Regional comparison showed the best correlation (r = 0.76, n = 45, p < 0.001) occurred inferior to the optic disc. Less positive but still highly significant correlations were seen superiorly and temporally (r = 0.52, n = 26, p = 0.007 and r = 0.49, n = 86, p = < 0.001 respectively), with the lowest correlation occurring at the nasal aspect of the disc (r = 0.06, n = 67, p = 0.64). In the primate eye, retinal nerve fibre layer thickness shows a positive correlation with retardation measurements obtained with the nerve fibre analyser. However, since the correlation coefficient varied around the optic disc, further evaluation of the device is advised before its routine clinical use.