Citrus fruits are one of the most important fruits in the global food industry due to their unique taste and nutritional benefits. Herein, we characterize the physicochemical and bioactive attributes of twenty-nine Greek citrus accessions, including oranges, mandarins/clementines, lemons, bergamot, citrons and lime along with twenty-seven highly commercial international cultivars. The assessed genotypes differ in various quality traits including color, ripening, and textural attributes. Several indigenous cultivars displayed desirable organoleptic traits, such as the oranges 'Valencia Oval Porou' (e.g., juice content and ascorbic acid) and 'Sanguine Gouritis' (eg., soluble solids (SSC) and acidity (TA) ratio), the mandarin 'Clementine Porou' (e.g., SSC/TA) and the lemon 'Vakalou' (e.g., firmness, acidity). Differences in primary metabolites, mainly in sugars, organic acids and amino acids were recorded among the tested species and cultivars. In addition, the autochthonous orange cultivars 'Sanguine Gouritsis' and 'Valencia Oval Porou' contained high sucrose levels whereas 'Lainato Chanion' had high hesperidin content. This large-scale analysis supports the ample availability of genetic resources for the development of citrus cultivars with improved nutritional quality traits.