The intramuscular fat (IMF), recognized by the consumer as marbling, is an important meat quality trait. The objective of this study was to validate a new method of beef marbling evaluation by image analysis. The new assessment was compared with two known marbling measurements: chemical IMF and USDA scores. Moreover, the relationship between marbling measurements and other meat quality parameters was investigated. Samples of Longissimus thoracis et lumborum (LTL) muscle were obtained from carcasses of 39 Italian crossbred heifers and 62 Angus bred heifers, that underwent two different dietary treatments. The use of various breeds and diets was chosen to obtain different intramuscular fat levels, in order to validate the use of Image J software for the analysis of different type of beef meat. The images of fresh cuts were appraised by experienced beef graders, and the samples were used to determine fat content by chemical Soxhlet extraction. Carcasses measurements according to the EUROP system, and other physical meat proprieties were also assessed. The results demonstrated that the marbling measurements obtained by computer image analysis, such as the number of marbling particles, the average particle size (mm), and the percentage of marbling particles (%), significantly (P < 0.05) correlated with USDA scores and IMF content. Moreover, the principal component analysis (PCA) showed three principal meat components, identified as 1) color, 2) fat, and 3) water release. The second principal component (PC) explained 24.94% of variance, and was positively correlated with image analysis measures, USDA score, and IMF, while negatively correlated with the Warner–Bratzler shear force (WBSF).