Grass pollen is the main cause of hay fever and allergic asthma in warm temperate climates during summer. The aim of this study was to determine the content of group 5 major allergens in pollen grains of agriculturally important grass species/cultivars. For each cultivar flowering dates and pollen production of cut anthers were observed in the field and in a climate chamber, respectively. An ELISA was used to quantify the group 5 allergens (Phl p5) in pollen extracts which were gained from the grass species Kentucky bluegrass, perennial rye grass, timothy, cocksfoot, annual / Italian rye grass, hybrid rye grass and festulolium. The group 5 allergen content of species varied between 0.01 ng (Kentucky bluegrass) and 0.06 ng (timothy) per pollen grain. On cultivar level the pollen allergenic content differed up to 74-times within the selected grass species. Results from this study might be helpful for the reduction of allergen exposure coming from agriculture grass production e.g. by an adapted grass selection or by the cultivation of grasses with low allergenic content in plant breeding.