The amount of free glucose and sucrose within the leaves of Stevia rebaudiana has been evaluated under different photoperiods and related to steviol glycoside (SVgly) accumulation. The diurnal dynamics of glucose and sucrose in leaves, internodes, apices, and roots revealed considerable fluctuations. Within leaves, glucose and sucrose content decreased up to three-fold during the night whereas SVgly amounts did not show any significant diurnal fluctuations. Ontogenetic variations in glucose content was markedly different under long-day (LD) and short-day (SD) regimes. Under LD, glucose content increased two-fold after the onset of flower bud formation, whereas under SD, a stagnation or small decrease was observed. This increase under LD was already noticed in the upper sink leaves at the end of the vegetative stage, possibly acting as a metabolic trigger for the phase transition to reproductive development. Under SD, glucose content appeared more controlled resulting in a better linear relationship with dry matter and SVgly content. The increased sink demands of the transitional apex led to a rapid decline in leaf sucrose which showed little or no correlation with SVgly amounts. From our results, it becomes clear that the correlation between glucose or sucrose as substrates and SVglys as products is very dynamic and is significantly influenced by day-length and ontogeny.