Ten Italian globe artichoke clones belonging to the Romanesco typology were characterized in the western coastal area of Italy (Cerveteri, Rome), using a combination of morphological (UPOV descriptors), biochemical (HPLC analysis), and molecular (AFLP, ISSR, and SSR markers) traits. Significant differences among clones were found for many of the quantitative and qualitative morphological traits. Multivariate analyses (Principal Component Analysis) showed that, of the 47 morphological descriptors assessed, four (i.e., plant height, central flower-head weight, earliness, and total flower-head weight) presented a clear grouping of the clones. Biochemical analyses showed that the clones significantly differed in the polyphenolic profiles of the flower-head, with the suggestion that some of these, such as S2, S3, S5, and S18, are more suitable for the fresh market. The clones, clustered by a UPGMA dendrogram based on 393 polymorphic AFLP and ISSR loci, showed that the clones were genetically separated from each other. This highlights the importance of characterizing, evaluating, and conserving autochthonous germplasm for future plant breeding activities. Overall, these studies resulted in the identification of two new clones, selected on the basis of flower-head morphology and earliness. These clones, named Michelangelo and Raffaello, are registered on the Italian National Register of Varieties (DM n. 6135, 3/29/2013 G.U. 91, 18 April 2013).