Key nutritional characteristics of the fruit flesh of 41 sour cherries growing in the region of Umbria in central Italy have been determined. Fruit size, flesh dry matter content, nonstructural carbohydrates, organic acids, and anthocyanins were the analyzed parameters. Both the growing environment and genotype were statistically significant for most of the characteristics. Morello sour cherries were characterized by a large amount of sorbitol (up to 44.2 mg·g−1 FW), which contributed significantly to the dry matter content of the flesh, malic acid content that was higher (up to 48.4 mg·g−1 FW) than any published values for cherry flesh, and high anthocyanin content (up to 383.4 mg per 100 g FW). Cyanidin 3-glucosyl rutinoside was the most abundant compound. The analyzed germplasm could be the basis for breeding programs and new industrial products with high nutritional value.