A total of 19 sesquiterpene lactones were isolated from roots of Lactuca canadensis L., of which 10 were reported for the first time from Lactuca species and two were unknown. This is also the first report on the co-occurrence of three pairs of zaluzanin C-type guaianolides, epimeric at C-3, and on the presence of six eudesmanolides, oxygenated at C-1 and C-3, in Lactuca species. The new compounds were characterized as 3-epizaluzanin C-3- O

b -glucopyranoside and 11,13-dehydrolactuside C using 1D and 2D NMR and high resolution mass spectroscopy. The sesquiterpene lactone profile of this species is dominated by zaluzanin C-type guaianolides (9 compounds) and eudesmanolides (8 compounds). The dissimilarity of this profile compared to that of other taxa of the genus is discussed.