Aim. To investigate the peculiarities of the functioning of the photosynthetic apparatus of plants of Carlina L. species under different light conditions of in vitro cultivation and under natural conditions using the method of induction of fluorescence of chlorophyll a. Methods. Methods of in vitro plants cultivation, a method of inducing fluorescence of chlorophyll a in light-adapted leaves. Results. It has been demonstrated that in vitro conditions reflect the evolved light growth requirements of species. Our previously obtained results regarding a closer match of physiological needs of in vitro plants of the species of Carlina onopordifolia Besser ex Szafer, Kulcz. et Pawt. to light conditions of variant 1.1 have been confirmed. Based on the synthesis of the dynamics of photosynthetic pigment content and their ratios in in vitro plants of Carlina acaulis L., as well as key parameters of fluorescence under different cultivation light conditions, a conclusion has been drawn about the greater alignment of variant 1.1 with the needs of plants of this species. It is hypothesized that in natural conditions, plants of the species of C. onopordifolia and Carlina cirsioides Klokov undergo abiotic stresses, manifested in increased dissipation of light energy into heat and the processes of photo-inhibition. There is significant enhancement in electron transport within the light-harvesting complex and reduction in the efficiency of quantum yield and light energy storage by Photosystem II. The vitality index of these species in their natural growth conditions is found to be half of the commonly accepted optimum value and 1.97–2.96 times lower compared to experimental in vitro plant groups. Conclusions. The obtained results suggest the usefulness of using the chlorophyll fluorescence induction method for assessing the functioning of the photosynthetic apparatus of in vitro plants of the genus Carlina.