AIM: To assess the reproducibility of Cirrus high-definition optical coherence tomography (HD-OCT; Carl Zeiss Meditec, Dublin, CA, USA) for analysis of peripapillary retinal nerve fiber layer (RNFL) thickness in glaucomatous eyes METHODS: Forty-five eyes (one eye from each glaucomatous patient) were imaged with Cirrus HD-OCT. Each eye was imaged three times by two separate operators. Intraclass correlation coefficient (ICC), coefficient of variation (CV), and test-retest variability were evaluated for both intraobserver and interobsever measurements RESULTS: In intraobserver measurements, the average RNFL thickness ICC was 0.983. CV and test-retest variability were 2.3% and 4.4 μm respectively. In quadrants ICC ranged from 0.886 to 0.956, the lowest associated with nasal quadrant and CV ranged from 3.6% to 7.7%. In interobsever measurements, the average RNFL thickness ICC was 0.979. CV and test-retest variability were 2.4% and 4.5 μm respectively. In quadrants ICC ranged from 0.886 to 0.957, the lowest associated with nasal quadrant and CV ranged from 3.8% to 8.6%. CONCLUSION: The reproducibility of Cirrus OCT for RNFL thickness is sufficiently good to be useful clinically as a measure of glaucoma progression.