The nutritional composition, polyphenol and anthocyanin composition, and antioxidant capacity of 52 colored highland barley were evaluated. The results showed that the protein content of highland barley in the black group was the highest, the total starch and fat contents in the blue group were the highest, the amylose content in the purple group was quite high, the fiber content in the yellow group was quite high, and the β-glucan content of the dark highland barley (purple, blue and black) was quite high. The polyphenol content and its antioxidant capacity in the black group were the highest, while the anthocyanin content and its antioxidant capacity in the purple highland barley were the highest. Ten types of monomeric phenolic substances were the main contributors to DPPH, ABTS, and FRAP antioxidant capacity. All varieties could be divided into four categories according to nutrition or function. The grain color could not be used as an absolute index to evaluate the quality of highland barley, and the important influence of variety on the quality of highland barley also needed to be considered. In actual production, suitable raw materials must be selected according to the processing purpose and variety characteristics of highland barley.