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Strawberry Anthracnose Fruit Rot (AFR) is one of the main limiting factors for strawberry production industry worldwide including Iran. Due to the restrictions associated with fungicides application across strawberry fields, their adverse effects on environment and the possible fungicides resistance development among fungal strains of the pathogen, the use of resistant cultivars is considered the most effective method for the management of this disease. In this study, reactions of 25 commercial strawberry cultivars were evaluated against the fungus Colletotrichum nymphaeae, causing strawberry AFR using fruit, leaf, and crown-based assays. According to the results of this study, the strawberry cultivars showed different reactions to the disease depending on the inoculation of their leaf, fruit, and crown with C. nymphaeae PET1 under in vivo and greenhouse conditions. However, fruit-based assay was a better indicator of AFR disease susceptibility due to nature of AFR disease caused by C. nymphaeae. Overall, ‘Blakemore’ and ‘Kurdistan’ cultivars were significantly more resistant compared with others, except ‘Aliso’, ‘Mrak’, ‘Diamant’, ‘Yallova’, ‘New Kurdistan’, ‘Mac Donance’ and ‘Ten Beauty’, respectively. On the contrary, ‘Gaviota’ cultivar was significantly the most susceptible than the rest, except ‘Camarosa’. The common commercial cultivars grown in Iran including ‘Camarosa’, ‘Paros’, ‘Pajaro’, and ‘Queen Eliza’ were categorized on the list of susceptible and highly susceptible cultivars in this study. To the best of our knowledge, this is the first study evaluating the reaction of some commercial strawberry cultivars against C. nymphaeae causing strawberry AFR.