The effect of reduced availability of sugars on growth and essential metabolic processes in roots, resulting from decreased photosynthesis under salinity, was excluded by establishing a non-photosynthetic model-system in this study: root cultures of Centaurium maritimum (L.) Fritch and Centaurium spicatum (L.) Fritch. The contribution of inorganic cations and organic compounds (e.g. carbohydrates and amino acids) to the osmotic adjustment (OA) in roots during short-term exposure to various salt concentrations (0, 50, 100 or 200 mM NaCl) was emphasized. Observed morphological and histological changes in roots were species specific, and were dependent on salinity level. Although C. spicatum appears to be more tolerant to salt stress, both species employed similar strategies in response to elevated salinity to different extents, and displayed effective OA mechanisms. Under low and moderate salinity, inorganic cations were the major contributors to OA in roots of both species, followed by soluble sugars, while the relative contribution of proline (Pro) and free amino acids was insignificant. Osmotic adjustment under severe stress appears to be mediated by increased accumulation of organic compounds. The analysis of the intraspecies variability in salt response of C. spicatum and C. maritimum roots enabled the identification of some organic compounds which could be used as potential biochemical markers in screening for salt tolerance, including Pro in C. spicatum, and trehalose and polyols in C. maritimum.