Feverfew (Tanacetum parthenium) is a medicinal herb which is rich in sesquiterpene lactones. The main sesquiterpene lactone in feverfew is parthenolide. Costunolide has been considered to be the immediate precursor of parthenolide. Parthenolide and costunolide levels in flowers and in leaves collected during the vegetative and generative phase of three feverfew genotypes (diploid, tetraploid and Zardband cv.) and in 2,4-D treated leaves of the diploid genotype were analyzed using UPLC–MS/MS. Parthenolide and costunolide levels showed a similar pattern in all genotypes, and were higher in flowers than in leaves. The developmental pattern of these two compounds was not the same in flowers of the different genotypes and 2,4-D treatment increased both coordinately. In conclusion, we show that genetical, developmental and spatial factors all influence the composition of sesquiterpene lactones in feverfew