The pitch and orthogonality of two-dimensional (2D) gratings have been calibrated by using an optical diffractometer (OD) and a metrological atomic force microscope (MAFM). Gratings are commonly used as a magnification standard for a scanning probe microscope (SPM) and a scanning electron microscope (SEM). Thus, to establish the meter-traceability in nano-metrology using SPM/SEM, it is important to certify the pitch and orthogonality of 2D gratings accurately. ODs and MAFMs are generally used as effective metrological instruments for the calibration of gratings in nanometer range. Since two methods have different metrological characteristics, they give complementary information for each other. ODs can measure only mean pitch value of grating with very low uncertainty, but MAFMs can obtain individual pitch value and local profile as well as mean pitch value, although they have higher uncertainty. Two kinds of 2D gratings, each with the nominal pitch of 700 nm and 1000 nm, were measured, and the uncertainties of calibrated values were evaluated. We also investigated the contribution of each uncertainty source to the combined standard uncertainty, and discussed the causes of main ones. The expanded uncertainties (k = 2) of calibrated pitch values were less than 0.05 nm and 0.5 nm for the OD and the MAFM, and the calibration results were coincident with each other within the expanded uncertainty of the MAFM.