Nuclear ribosomal (ITS) DNA sequence data from representatives of eleven out of 12 genera previously included in Thlaspi sensu late were analyzed to elucidate relationships within the former genus Thlaspi sensu late. Sequences from Thlaspi segregates Noccaea, Masmenia, Callothlaspi, Kotschyella, Microthlaspi, and Noccidium were added to the existing data sets, and only some Thlaspi sensu late segregates formed a monophyletic group. The recently renamed genus Pseudosempervivum, formerly part of the genus Cochlearia, has been shown to be closely related to this group as well. Microthlaspi consists of three independent lineages of which Microthlaspi umbellatum is closely related to Neurotropis. The Thlaspi s.str. taxa including the type species T. arvense, are closely related to Peltaria and Alliaria and they represent a monophyletic group. Kotschyella and Noccidium, which were also integrated into Thlaspi s.l. are not closely related to any Thlaspi lineage. Therefore, we suggest major taxonomical changes which are not in agreement with concepts based on morphological data. Our ITS based phylogeny demonstrates that subtribe Thlaspidinae comprises some additional genera such as Teesdalia, Peltaria, and Alliaria, the latter two even previously classified in other tribes than tribe Lepidieae.