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Thapsia garganica L. and T. transtagana Brot. are classified as synonymous in Flora Europaea. In the present investigation significant differences between the two taxa with regard to fruit anatomy and phytochemistry are demonstrated and they are considered as two separate species. Microscopic studies revealed a distinct difference in number and position of secretory spaces in the pericarp of fruits from the two species and in addition pronounced differences were found in the presence of thapsigargins, the bioactive constituents of the two species. Quantitative HPLC analyses of thapsigargins were carried out on different plant organs from T. garganica and T. transtagana collected from various locations. Thapsigargin, thapsigargicin, nortrilobolid and thapsivillosin I and J were dominant compounds in all organs of T. garganica whereas none of these compounds could be detected in any organ of T. transtagana. On the contrary, thapsitranstagin and trilobolid were main thapsigargins of T. transtagana. T. garganica may include two chemotypes, as trilobolid was detected in some specimens of T. garganica only. As this is the first time trilobolid has been detected in these two species, it was isolated from both and identified by 1H NMR‐spectroscopy.