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Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops highly appreciated for the nutritional properties and content of beneficial compounds for human health. After its domestication, this crop has been spread throughout the world and found its secondary centre of diversification in the Mediterranean basin, where numerous landraces have been developed. Among these, 'San Marzano' is a famous and remunerative PDO variety for processing and fresh market consumption. 'Re Fiascone' is an ancient cultivar for which ongoing efforts aim to promote its diffusion and commercialisation. Both find their origin in the Campania region in Southern Italy where in the past decades, several accessions have been selected and handed down by farmers. This study reports a comprehensive assessment for morphological and biochemical traits of twenty-one accessions of the two landraces. Fruit morphology and content of sugars and flavonoids were the most discriminating parameters among cultivars. Among sugars and organic acids, fructose and citric acid were the most represented compounds, respectively. We found level of trans-lycopene and β-carotene up to 112.82 and 16.29 µg g−1 of fresh product, respectively, while ascorbic acid levels reached values up to 22.48 mg 100 g−1 of fresh weight. Molecular analysis has been performed using a double approach for microsatellite genotyping based on capillary electrophoresis and high-resolution melting. Results highlighted a separation of the accessions according to 'Re Fiascone' and 'San Marzano' identifying both unique and admixed accessions between the two groups. The study approach aims at the recovery and valorisation of local genetic resources, but also at the identification of traits of interest to transfer in breeding programs. [ABSTRACT FROM AUTHOR]