•Stevia strains can be grown in temperate climate under the tunnel and in the field.•Differences between stevia strains resulted from differences in metabolic activity.•High photosynthetic efficiency is correlated with high steviol glycosides level.•Steviol glycosides might function in the protection of photosynthetic apparatus. The objective of this study was to evaluate the development and the quality of leaves from several S. rebaudiana Bertoni strains cultivated under temperate climate conditions in the tunnel and in the open field in the relation to the photosynthetic activity. Investigated stevia strains differed significantly in terms of whole plant and leaf morphology, photosynthetic efficiency as well as in the content of steviol glycosides, and antioxidant capacity, independently of cultivation place. On the basis of several chlorophyll a fluorescence parameters analysis it was stated that plants of all examined strains, did not exhibit photosynthesis inhibition. The studies of the relationship between photosynthetic activity and the level of secondary metabolites indicated that the highest content of steviol glycosides and phenolic compounds had plants showing also the highest value of the energy fraction photochemically converted in PS II (YII), while the highest ratio of rebaudioside A to stevioside content corresponded with low amount of energy dissipated as a heat (YNPQ). This results might indicate that steviol glycosides play a role in the better adaptation to environmental conditions. Karyology and cytometric studies revealed that all investigated stevia strains were diploids without significant differences in 2C DNA amount. Therefore, observed diversity in the content of steviol glycosides and phenolic compounds between investigated S. rebaudiana strains was not the effect of polyploidization but probably resulted from the differences in the metabolic activity of the examined plants.