The site-specific natural hydrogen isotope ratios of plant metabolites determined by 2H nuclear magnetic resonance (SNIF-NMR method) can provide powerful criteria for inferring mechanistic and environmental effects on biosynthetic pathways. This work examines the potential of isotopic profiles for the main constituents of carbohydrates, glucose and fructose, to distinguish different photosynthetic pathways. An appropriate analytical strategy, involving three suitable isotopic probes, has been elaborated with a view to measuring simultaneously, in conditions devoid of isotopic perturbations, all (or nearly all) of the carbon-bound hydrogen isotope ratios. It is shown that the type of photosynthetic metabolism, either C3 (sugar beet, orange, and grape), C4 (maize and sugar cane), or CAM (pineapple), and the physiological status of the precursor plant exert strong influences on the deuterium distribution in the sugar molecules. Consequently, this isotopic fingerprint may be a rich source of information for the comparison of mechanisms in metabolic pathways. In addition, it can provide complementary criteria to ethanol as a probe for the origin of sugars.