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Proso millet has been proposed as an effective anti-diabetic food thanks to the combined action of polyphenols and starch. This study aimed to characterize the chemical composition of twenty-five accessions, in order to enhance this cereal as an alternative to available starch for food applications or to propose new food ingredients with health benefits. Proso millet contained a high percentage of starch, reaching values of 58.51%. The amylose content showed high variability, with values ranging from 1.36 to 42.70%, and significantly higher values were recorded for the white accessions than for the colored ones. High-resistant starch content (13.41-26.07%) was also found. The HPLC-MS analysis showed the same phenolic pattern in all the samples. Cinnamic acids are the most abundant compounds and significant differences in their total content were found (0.69 to 1.35 mg/g DW), while flavonoids were only detected in trace amounts. Statistical results showed significantly higher antiradical activity in the colored accessions than in the white ones.