In Apuglia, Southern Italy, the favorable climate conditions has allowed the diffusion of many unknown genotypes spread in different orchards and field collections. In this study, 19 SSR markers have been developed and characterized in Punica granatum. Eight selected loci were used to identify ten pomegranate accessions collected in different orchards in Apuglia region. Chemical-nutritional analyses (antioxidant activity, total phenolic, titrable acidity, pH and Brix) were also performed on a selected number of cultivars chosen on the basis of the results of DNA analysis. Results showed good levels of polymorphism and indicated a high antioxidant activity and total phenol content especially in the fruit of two accessions: Chionna and Cisternino.