To assess the agreement of central and paracentral corneal thickness measurements between ultrasound pachymetry (USP), Orbscan II, and Visante anterior segment optical coherence tomography (ASOCT). Observational cross-sectional study. Seventy eyes of 70 subjects. Each subject underwent Orbscan II (using an acoustic equivalent correction factor of 0.89), ASOCT, and USP examination. Bland-Altman plots were used to evaluate agreement between instruments. Central and paracentral corneal thickness measurements by the 3 methods and agreement, as evaluated by 95% limits of agreement (LOA). The mean measurements of average central corneal thickness by USP, Orbscan II, and ASOCT were 553.5+/-30.26 microm, 553.22+/-25.47 microm, and 538.79+/-26.22 microm, respectively. There was high correlation between instruments: USP with ASOCT (r = 0.936, P<0.001), USP with Orbscan II (r = 0.900, P<0.001) for central corneal thickness measurements, and Orbscan II with ASOCT for average paracentral 2- to 5-mm measurements (r = 0.947, P<0.001). The mean differences (and upper/lower LOA) for central corneal thickness measurements were 0.31+/-13.34 microm (26.44/-25.83) between USP and Orbscan II, 14.74+/-10.84 microm (36.0/-6.51) between USP and ASOCT, and 14.44+/-9.14 microm (32.36/-3.48) between Orbscan II and ASOCT. The average mean difference (and upper/lower LOA) between Orbscan II and ASOCT for paracentral 2- to 5-mm corneal thickness measurements was 10.35+/-8.67 microm (27.35/-6.65). Anterior segment optical coherence tomography underestimated corneal thickness compared with that measured with USP. Anterior segment optical coherence tomography had better agreement with the gold standard USP, as compared with Orbscan II. However, important discrepancies among instruments exist. Clinicians should be aware that corneal thickness measurements are influenced by the method of measurement and that, although highly correlated, these instruments should not be used interchangeably for the assessment of corneal thickness.