This study deals with the qualitative characterization of the phenolic profile of pomegranate juices obtained from ancient accessions. Composition data, together with genetic, morphological, and agronomical parameters, may lead to a full characterization of such germplasm, with the aim of its retrieval and biodiversity valorization. Environmental adaptation, indeed, may contribute to an enrichment of the phenolic content in pomegranate, with important effects on its nutritional properties. More than 65 punicalagins, ellagic acid derivatives, flavonoids, anthocyanins, and phenylpropanoids were simultaneously detected from four centuries old Punica granatum L. ecotypes from northern Italy and compared with those of P. granatum cv. Dente di Cavallo, a widely cultivated Italian cultivar, using a simple ultra-HPLC (uHPLC) separation and MS(n) linear ion trap mass spectrometric characterization. Fingerprinting phytochemical discrimination of the accessions was obtained by chemometric analysis despite their limited geographical distribution, confirming the great intraspecific variability in pomegranate secondary metabolism. The combined recourse to uHPLC-MS(n) qualitative fingerprinting and multivariate analysis may represent a useful tool for the discrimination and selection of pomegranate germplasm with specific properties related to polyphenolic content.