Characterization of local germplasm is an effective way to identify elite breeding material and develop improved varieties. This study was aimed to assess 52 tomato accessions comprised of local varieties (28), landraces (8), breeding lines (14), and wild relatives (2), and their characterization for 30 morphological/agronomic, four fruit quality, and tomato mosaic virus (ToMV) resistance traits. Morphological, quality, and ToMV traits were evaluated using phenotyping, biochemical assays, and molecular markers, respectively. Fruit shape and size showed appreciable variation, with fruits varying from rounded to heart shaped and small to big size. Significant variation was observed for fruit weight (1.6–564.8 g), fruits per plant (6.0–174.7), productivity (130.5–5146.5 g), soluble solids (4.1%–8.4%), vitamin C (9.5–46.4 mg·100 g−1), antioxidant activity (2.5–9.6 μmol Fe2+·g−1fresh weight), and total polyphenols (23.9–124.2 GAE·100 g−1fresh weight). All accessions were phenotypically screened for the virus resistance in the growth chamber, and CAPS molecular markers were used to identify accessions with ToMV Tm-22resistant alleles, and accessions LYC-13, LYC-15, LYC-17, LYC-26, and LYC-52 were identified as resistant. Multivariate analysis of morphological and quality traits showed that 35 principal components contributed to the total variation and the first two and 12 principal components explained 47.2% and 90% of the variation, respectively. The evaluated tomato collection appears to have breeding potential, and around 20% of the accessions in the collection (LYC-6, LYC-17, LYC-18, LYC-26 to LYC-31, and LYC 33) are promising genetic resources for variety development that are enriched with enhanced fruit quality and high yield.