Hawthorn (Crataegus spp.) berry has long been used in traditional Chinese medicine and as a functional food. In order to investigate the variation in metabolites in different colored hawthorn berry varieties and to identify the constituents responsible for their color trait and antioxidant activity, four different-colored hawthorn berries were analyzed and compared using LC-MS/MS based on comparative metabolomic analysis. A total of 728 metabolites were identified. The metabolic compounds differed among the samples. The differentially activated metabolic pathways included phenylpropanoid biosynthesis, phenylalanine metabolism, flavone and flavonol biosynthesis, and amino acids biosynthesis. The hawthorn berry color was caused mainly by a combination of five anthocyanins, pH (influenced by organic acids), and non-anthocyanin flavonoids present as co-pigments. Polyphenol compounds, including phenolic acids and flavonoids, were closely associated with antioxidant activity in hawthorn berry. In addition, significantly higher (P < 0.05) antioxidant activity was found in the dark red sample variety (C. pinnatifida Wulingzirou, WLZR), and may be related to the high concentration of 32 polyphenols. These findings provide valuable insight into the metabolite composition, function and the mechanism of color formation in hawthorn berry.