A selection of tomato (Solanum lycopersicum L.) genotypes with diverse origin and breeding history including 33 landraces, eight modern varieties and two commercial hybrids has been characterized using a set of 25 qualitative descriptors and six quantitative traits. A wide range of variation was evident for the majority of traits, highlighting their utility for characterizing tomato germplasm collections. A plethora of qualitative traits including type of leaf blade, depression and ribbing at peduncle end, fruit shape at blossom end, number of locules and flowering time, as well as measured traits with economic importance such as fruit fresh weight, firmness and total yield per plant, were found to be highly variable within the collection, with a diversity index greater than 0.8. Strong correlations were detected among several traits related to fruit yield and quality. Two-dimensional principal component analysis as well as the unsupervised hierarchical clustering grouped genotypes according to their phenotypic resemblance and morphological characteristics to a great extent. Landraces from different origins were scattered across the whole variation spectrum of PC1 and PC2. A set of six qualitative traits could efficiently discriminate cultivars in PCA (explaining 75% of total variation), suggesting that it can serve as a valuable breeding tool for the germplasm characterization. The evaluation of the phenotypic diversity in the collection as well as the identification of traits that contribute most to heterogeneity have important implications for establishing core collections with high diversity, as well as designing breeding schemes across the Mediterranean basin.