P>Background: To evaluate the agreement and repeatability between operator-dependent centring and automatic centring retinal nerve fibre layer thickness measurement patterns (RNFL3.45 and NHM4) of RTvue optical coherence tomography in normal and glaucomatous eyes. Design: Cross-sectional observational study. Participants: A total of 153 eyes from 149 normal subjects and subjects with glaucoma were analysed. Methods: The retinal nerve fibre layer thickness was measured using RNFL3.45 and NHM4 three times on the same day to determine the repeatability and agreement between the two scan patterns. Main Outcome Measures: Student's paired t-testing, intra-class correlation coefficient, coefficient of variation, test-retest viability, Pearson's correlation coefficient and Bland-Altman analysis of retinal nerve fibre layer thickness measurements. Results: The difference between RNFL3.45 and NHM4 measurements was statistically significant by paired t-testing (P = 0.003) only in severe glaucoma group. The Pearson's correlation test showed a high degree of correlation of the mean retinal nerve fibre layer thickness (r = 0.949). Bland-Altman plots showed that the differences between RNFL3.45 and NHM4 were smaller at thicker retinal nerve fibre layer values, but larger at thinner retinal nerve fibre layer values. The intra-class correlation coefficient for RNFL3.45 (and lower 95% confidence interval) in normal and glaucomatous eyes was 0.990 (0.985 confidence interval) and 0.997 (0.995 confidence interval), respectively. The intra-class correlation coefficient for NHM4 in normal and glaucomatous eyes was 0.989 (0.983 confidence interval) and 0.995 (0.992 confidence interval), respectively. The test-retest variability for RNFL3.45 and NHM4 ranged from 3.98 to 9.75 mu m in normal eyes, and from 4.10 to 13.34 mu m in glaucomatous eyes. Conclusion: Measurements of retinal nerve fibre layer thickness by RNFL3.45 and NHM4 are in good agreement.