Objective To measure and compare retinal nerve fiber layer (RNFL) thickness by optical coherence tomography (OCT) in normal eyes, ocular hypertensive eyes, and primary open-angle glaucoma patients. Methods The RNFL thickness was measured in 3 groups: normal group (76 cases, 152 eyes), ocular hypertensive group (70 cases, 109 eyes), and primary open-angle glaucoma group (67 cases, 112 eyes). RNFL measurements were taken with OCT CIRRUS Zeiss. Results The average values of RNFL thickness were (107.61±8.03) μm, (105.26±9.22) μm, and (91.00±10.55) μm in normal eyes, ocular hypertensive eyes, and glaucomatous eyes, respectively. Compared normal eyes, RNFL average thickness, superior thickness, and inferior thickness showed significant differences in glaucomatous eyes (P<0.01). The RNFL average thickness was significantly different between ocular hypertensive eyes and glaucomatous eyes (P<0.01), whereas there was no significant difference between normal and ocular hypertensive eyes (P>0.05). Conclusion The RNFL thickness measurement is a kind of very useful and objective method to differentiate ocular hypertensive and primary open-angle glaucoma.