We characterized atherosclerotic plaque components with a novel cryo-imaging system in lieu of standard histological methods commonly used for imaging validation and research endpoints. We aim to accurately identify plaque tissue types from fresh cadaver specimens rapidly (less than 5 h) in three dimensions for large specimens (up to 4 cm vessel segments). A single-blind validation study was designed to determine sensitivity, specificity and inter-rater agreement (Fleiss' Kappa) of cryo-imaging tissue types with histology as the gold standard. Six naïve human raters identified 344 tissue type samples in 36 cryo-image sets after being trained. Tissue type sensitivities are as follows: greater than 90% for adventitia, media-related, smooth muscle cell ingrowth, external elastic lamina, internal elastic lamina, fibrosis, dense calcification and haemorrhage; greater than 80% for lipid and light calcification; and greater than 50% for cholesterol clefts. Specificities were greater than 95% for all tissue types. The results demonstrate convincingly that cryo-imaging can be used to accurately identify most tissue types. If the cryo-imaging data are entered into visualization software, three-dimensional renderings of the plaque can be generated to visualize and quantify plaque components.