In photosynthetic eukaryotes, there are two well-characterized fructose-1,6-bisphosphatases (FBPases):the redox-insensitive cytosolic FBPase (cyFBPase), which participates in gluconeogenesis, and the redox-sensitive chloroplastic FBPase (cpFBPase1), which is a critical enzyme in the Calvin cycle. Recent studies have identified a new chloroplastic FBPase, cpFBPase2; however, its phylogenetic distribution, evolu-tionary origin, and physiological function remain unclear. In this study, we identified and characterized these three FBPase isoforms in diverse, representative photosynthetic lineages and analyzed their phy-logeny. In contrast to previous hypotheses, we found that cpFBPase2 is ubiquitous in photosynthetic eukaryotes. Additionally, all cpFBPase2s from diverse lineages form a monophyly, suggesting cpFBPase2 is not a recently evolved enzyme restricted to land plants but rather evolved early in the evolution of photosynthetic organisms, and most likely, in the common ancestor of photosynthetic eukaryotes. cyFBPase was probably first duplicated to produce cpFBPase2, and then the latter duplicated to produce cpFBPase1. The ubiquitous coexistence of these two cpFBPases in chloroplasts is most likely the conse-quence of adaptation to different redox conditions of photosynthesis, especially those caused by recur-rent changes in light conditions.