Species of the genus Isoplexis are of particular interest with respect to the biochemical pathway leading to the cardenolides. It is important to determine whether or not 5beta-configured compounds, typically produced by Digitalis species and used in medicine, are present together with their respective alpha-isomers in Isoplexis spp. Structure elucidation by LC-NMR of the products isolated from in vitro regenerated Isoplexis canariensis, I. chalcantha and I. isabelliana was carried out, and similarities were observed among the products of the three species, including the presence of digitoxigenin-type cardenolides in I. canariensis and xysmalogenin and canarigenin derivatives in I. chalcantha never previously reported in these species. Pregnane glycosides not found until now either in Isoplexis or Digitalis were also detected.