Purpose To compare the diagnostic ability of the Heidelberg Retina Tomograph 3 (HRT3) and the Heidelberg Retina Tomograph 2 (HRT2) for discriminating between healthy eyes and eyes with glaucomatous visual field loss. Design Retrospective cross-sectional study. Methods Participants were 93 healthy subjects and 90 patients with open-angle glaucoma. All participants underwent imaging of the optic nerve head with the HRT2. Afterward, HRT data also were analyzed using version 3 of the software without modifying the optic disk contour line. The receiver operating characteristic (ROC) curves between normal and glaucomatous subjects were plotted for the global stereometric parameters of both software versions. Moorfields regression analysis (MRA) and glaucoma probability score (GPS) diagnostic abilities also were compared. Results The parameters with the largest areas under the ROC curve were the Frederick S. Mikelberg (FSM) discriminant function for the HRT3 (0.948) and the vertical cup-to-disk ratio (0.914) for the HRT2. At a fixed specificity of 95%, the best sensitivity was 74.4% for the Reinhard O.W. Burk (RB) discriminant function of the HRT2 and 83.3% for the FSM discriminant function of the HRT3. The best sensitivity and specificity pairs for the HRT classifications were 85.5% and 76.3%, respectively, for overall MRA2, 84.4% and 83.8%, respectively, for overall MRA3, 93.3% and 58.0%, respectively, for the global color-coded GPS, and 84.4% and 74.1%, respectively, for the global GPS numerical value. Conclusions At 95% fixed specificity, most HRT3 parameters exhibited at least the same sensitivity for glaucoma diagnosis as the analogous parameters for the HRT2. The diagnostic ability overall of MRA3 was similar to that of the previous version. GPS exhibited higher sensitivity and somewhat lower specificity than the MRA.