© MYU K.K. Glycogen and moisture contents are associated with palatability, especially the flavor of Japanese wagyu beef samples. In this study, near-infrared (NIR) absorbance spectroscopy (12500–4000 cm−1) was carried out to predict the glycogen and moisture contents in Japanese wagyu beef. Calibration and prediction models were established by partial least-squares regression (PLSR) between the measured reference glycogen and moisture contents and the spectral data. Different spectral preprocessing methods were used, and the loading coefficient obtained from PLSR models was employed to select feature spectra. As a result, the prediction model of glycogen with the selected spectral region of 9000–4300 cm−1 after smoothing-standard normalized variate (SNV) yielded optimum results with a determination coefficient (Rp2) of 0.415, a root-mean-squared error of prediction (RMSEP) set of 0.386 mg/g, and a ratio of prediction to deviation (RPD) of 1.218. In addition, the prediction model of moisture content in the full spectral region of 12500–4000 cm−1 after smoothing-multiplicative scatter correction (MSC) yielded optimum results with Rp2 of 0.795, RMSEP of 2.669%, and RPD of 2.008. The results of this study demonstrated that NIR spectroscopy offers great potential for the prediction of glycogen and moisture contents in Japanese wagyu beef samples.