Sphaerophysa kotschyana is a threatened endemic species in Turkey and according to the Bern Convention, it is on the absolute preservation plant list. In vitro propagation methodologies were evaluated as an ex situ conservation strategy for this species. Nodal segments were cultured on Murashige and Skoog (MS) media with different cytokinins (benzyladenine, thidiazuron (TDZ) and zeatine), with or without auxin (a-naphthaleneacetic acid; NAA), to investigate shoot initiation. TDZ produced the highest number of shoots (11.0 shoots per explant) on MS medium at a concentration of 0.05 mg L-1. Rooting reached 100% when 0.5 mg L-1 NAA was combined with half strength MS and 1.5% sucrose and rooted plantlets were successfully acclimatised. Somaclonal variation of a mother plant and 10 regenerants was assessed using ISSR analysis. The same banding profiles were exhibited by all plants. In vitro response to salinity stress (NaCl) was also investigated in this halophytic species. Higher concentrations of NaCl negatively affected shoot multiplication, whereas shoot height was enhanced at 50mM NaCl. These results suggest that the established protocol is an efficient and reliable system of in vitro propagation for ex situ conservation of S. kotschyana.