Utilization of biomass is essential for reducing greenhouse gas emissions. It is therefore important to search for land and water-use efficient energy crops that can avoid diversion of arable land used for food crops. In this respect, desert plants could become potential crops for renewable energy production by anaerobic digestion as well as production of valuable compounds. The aim of this paper was to investigate the specific methane production as well as the triterpenoid biosynthesis, accumulation, and diversity of whole Aloe vera (L.) Burm.f. leaves and Opuntia robusta J.C. Wendl. cladodes. These plants were readily transformed by anaerobic digestion reporting cumulative biogas volumes of 3310.49 +/- 82.67 and 3685.19 +/- 207.74 NmL for A. vera and O. robusta, respectively. However, both substrates proved detrimental for the methanogenesis phase of anaerobic digestion giving specific methane production of only 8.22 +/- 0.09 and 28.88 +/- 2.63 NmL/g VS for A. vera and O. robusta, respectively. The observed inhibitory activity could be related to the high concentration of total triterpenoids in both substrates, 1.87 + 0.05 and 1.05 + 0.33 mu g/mg of DW for A. vera and O. robusta, respectively; these were the highest triterpenoid yields reported for species of the Aloe and Opuntia genera. Therefore, the triterpenoid biosynthesis pathways in these plants were explored. The presence of lupeol is reported for the first time in the Opuntia genus and a fragment from a putative lupeol synthase in O. robusta. Finally, the cycloartenol synthase of A. vera was functionally characterized and uncharacterized oxidosqualene cyclases of A. vera and O. robusta are reported. The results in this paper provide insights showing the potential of discovering new triterpenoids through further exploration of triterpenoid biosynthesis in these species.