The cross section images of beef rib-eye in spectral region of 400～1000nm were collected by using a hyperspectral imaging system. With comparative analysis of the spectral curves of the fat regions and the muscle ones of beef rib-eye images, it was found that the reflection intensity of fat regions and muscles areas for beef rib eye images in different spectral bands were obviously different. Fitting the reflection intensity ratio of the fat regions and muscle regions with the wavelength, it could be concluded that, in the 534nm band, the difference between the reflection intensity of fat regions and the one of muscle regions was the maximum. The image processings such as median filtering, region growing and Otsu’s thresholding etc. were conducted on the original color beef rib-eye images and the 534nm band beef rib-eye images for the segmentation of the beef marbling, respectively. The result showed that the higher segmentation accuracy of beef marbling could be obtained by using the 534nm band beef rib-eye images compared with the original color beef rib-eye images.