Salinity is one of reasons causing the greatest yield-reducing factors leads various physiological and biochemical effects. Recently, long non-coding RNAs (lncRNAs) became prominent as crucial regulators in various biological processes, including developmental processes and stress responses such as salinity. In this study, physiological responses of four Hordeum vulgare L. cvs genotypes (Beyşehir 99, Hasat, Konevi 98 and Tarm 92) were assessed for 150 mM salinity treatment for 3 days germination period, maize (CNT0018772) and rice (CNT0031477) lncRNAs localizations in barley chromosomes via fluorescence in situ hybridization (FISH). Physiological responses of barley varieties, including root and shoot heights, fresh and dry weights, water content (WC) and protein content were affected differently by 150 mM salinity stress. The germination percentage of Beyşehir 99 and Tarm 92 reduced while Konevi 98 was slightly increased. However, proline content changed only in Beyşehir 99. According to FISH results, the localizations of CNT0018772 and CNT0031477 were revealed up 6 and 8 signals, respectively. This study may contribute new insight into lncRNAs functions underlying the salinity tolerance mechanisms in different barley varieties.