In this paper, we discuss and utilize the multiple-reflection attenuated total reflectance (ATR) method of near-infrared spectroscopy (NIRS) for the determination of glycogen content in Japanese wagyu beef. We have also conducted experiments on diffuse reflection to compare the experimental results. Glycogen, as a reservoir of glucose units, has a positive effect on the palatability of wagyu beef. The obtained nontreated spectra were preprocessed by several different methods, then a calibration and prediction model between spectral data and glycogen content was developed by partial least squares regression (PLSR). We found that when the multiple-reflection ATR method and the diffuse reflection method are used for NIR spectroscopy methods, there is a significant difference in their ability to predict the glycogen content in wagyu beef. The determination coefficient of the best prediction model for the spectra obtained by the multiple-reflection ATR method was 0.72 for the measurement of glycogen content in wagyu beef, whereas that obtained by the diffuse reflection method was only 0.29. Through experiments and data analysis, we found that the multiple-reflection ATR method is promising for predicting glycogen content in wagyu beef. The results of these experiments have important implications for the development of future small portable beef glycogen sensors.