The pitch and orthogonality of two-dimensional (2D) gratings have been measured by using a metrological atomic force microscope (MAFM) and measurement uncertainty has been analyzed. Gratings are typical standard artifacts for the calibration of precision microscopes. Since the magnification and orthogonality in two perpendicular axes of microscopes can be calibrated simultaneously using 2D gratings, it is important to certify the pitch and orthogonality of 2D gratings accurately for nano-metrology using precision microscopes. In the measurement of 2D gratings, the MAFM can be used effectively for its nanometric resolution and uncertainty, but a new measurement scheme was required to overcome some limitations of current MAFM such as nonnegligible thermal drift and slow scan speed. Two kinds of 2D gratings, each with the nominal pitch of 300 nm and 1000 nm, were measured using line scans for the pitch measurement of each direction. The expanded uncertainties (k = 2) of measured pitch values were less than 0.2 nm and 0.4 nm for each specimen, and those of measured orthogonality were less than 0.09 degree and 0.05 degree respectively. The experimental results measured using the MAFM and optical diffractometer were coincident with each other within the expanded uncertainty of the MAFM. As a future work, we also proposed another scheme for the measurements of 2D gratings to increase the accuracy of calculated peak positions.