Crassulacean acid metabolism (CAM) provides desert plants with distinct advantages over the C3 and C4 photosynthetic pathways in harsh climates where water is scarce. CAM is, however more metabolically costly than C3 or C4 photosynthesis, and some plants, such as Mesembryanthemum crystallinum, facultatively utilize CAM when water is abundant, and water conservation unnecessary. In such situations, these plants behave akin to a C3 plant when photosynthesizing. CAM is divided into four phases, with each phase displaying unique metabolic processes. Certain changes, including changes in the timing of CO2 fixation, stable carbon isotope ratios, and tissue malic acid content accumulation patterns can indicate that a plant has shifted from CAM to C3 photosynthesis. Such shifts have been observed to be regulated primarily by water availability and ontogenic development. While facultative CAM is well documented in species like Mesembryanthemum crystallinum, and it has not been studied extensively in Agave with the exception of Agave deserti, and Agave angustifolia. A better understanding of this phenomenon would apply to the agricultural growth of this genus. This study aimed to trigger C3 to CAM shifts in Agave sensu lato species, in order to expand upon the findings of previous studies, and better understand the prevalence of facultative CAM expression in the genus. Gas exchange and stable carbon isotope measurements were taken from 2-month-old, 10-month-old, and mature agaves grown in controlled ocnditions. Tissue acid content measurements were taken from mature plants. Despite the Agave sensu lato species in this study being subjected to moisture applications ranging from dry to saturated, we were unable to observe any distinct shifts from CAM to C3 photosynthesis in any of the species tested for both seedlings and mature plants. Diel net CO2 fixation rates also increased with age, and water applications for seedlings, and decreased with heavy irrigation in mature plants. Stable carbon isotope ratios revealed that some carbon in the plant tissues was fixed by rubisco, and that some species (Polianthes tuberosa, Prochnyanthes mexicana) had carbon isotope ratios of a C3 plant, but these ratios did not change with different irrigation treatments. Malic acid accumulation remained typical of CAM plants for the species tested as well, with one exception in Polianthes tuberosa. As such, we conclude that the Agave sensu stricto species tested in this study are obligate CAM plants, and that they perform poorly mature individuals are over-watered. Additionally, the Agave sensu lato species P. mexicana, and P. tuberosa appear to be C3 plants given the results of this study.