We measured grating pitch standards using optical diffractometry and analyzed measurement uncertainty. Grating pitch standards have been used widely as a magnification standard for a scanning electron microscope (SEM) and a scanning probe microscope (SPM). Thus, to establish the meter-traceability in nano-metrology using SPM and SEM, it is important to certify grating pitch standards accurately. The optical diffractometer consists of two laser sources, argon ion laser (488 nm) and He-Cd laser (325 nm), optics to make an incident beam, a precision rotary table and a quadrant photo-diode to detect the position of diffraction beam. The precision rotary table incorporates a calibrated angle encoder, enabling the precise and accurate measurement of diffraction angle. Applying the measured diffraction angle to the grating equation, the mean pitch of grating specimen can be obtained very accurately. The pitch and orthogonality of two-dimensional grating pitch standards were measured, and the measurement uncertainty was analyzed according to the Guide to the Expression of Uncertainty in Measurement. The expanded uncertainties (k = 2) in pitch measurement were less than 0.015 nm and 0.03 nm for the specimen with the nominal pitch of 300 nm and 1000 nm. In the case of orthogonality measurement, the expanded uncertainties were less than $0.006^{circ}$. In the pitch measurement, the main uncertainty source was the variation of measured pitch values according to the diffraction order. The measurement results show that the optical diffractometry can be used as an effective calibration tool for grating pitch standards.