Tomatoes (Solanum lycopersicum) are one of the most consumed vegetables worldwide. However, tomato allergies in patients suffering from birch pollen allergy occur frequently. Due to highly similar protein structures of the tomato allergen Sola l 4 and the major birch pollen allergen Bet v 1, patients cross-react with allergenic proteins from tomato as well as other fruits or vegetables. The aim of this study was to quantify Sola l 4 in various tomatoes differing in color, size and shape for identification of varieties with a reduced allergen level. Therefore, an indirect competitive ELISA using a specific polyclonal Sola l 4 antibody was developed. In addition, two varieties, both cultivated either conventionally or organically and furthermore dried with different methods, were analyzed to investigate the influence of the cultivation method and processing techniques on Sola l 4 level. Within 23 varieties, Sola l 4 content varied significantly between 0.24 and 1.71 μg Sola l 4/g FW. The tomato cultivars Rugantino and Rhianna showed the significantly lowest level, whereas in cultivars Farbini and Bambello the significantly highest concentration was determined. Drying of tomatoes in the oven and by sun resulted in a significant decrease. The thermal instability was verified for the recombinant Sola l 4 emphasizing the results for the native protein in dried tomato samples. Overall, the Sola l 4 content is cultivar-dependent and no correlation between color and Sola l 4 amount was found. During the drying process of tomatoes Sola l 4 level was significantly reduced due to thermal instability. Growing conditions have a minor effect whereas seasonal effects show a more pronounced impact. These findings could extend the knowledge about the allergen level of different tomato varieties and may help to improve food safety to potentially increase the life quality of patients suffering from birch pollen allergy.