Lepidium draba, a medicinal herb belongs to the Brassicaceae family is more considered because it contains high level of glucoraphanin, as a precursor of sulforaphane (an anti-carcinogenic and anticancer agent). Whilst there has been studies on plant response to salinity stress, to date the response of L. draba to salinity stress is poorly understood. The aims of this study were Identification of Δ1- pyrroline-5-carboxylate synthetase (P5CS) gene and assessment of different concentrations of NaCl on P5CS gene expression level and some biochemical traits of L. draba treated seedlings. The five-day old L. draba seedlings were treated by different concentrations (0, 50, 100, 200 and 300 mM) of NaCl for 14 days in completely random design. Identification of the P5CS gene was done by purification of total RNA, synthesis of cDNA library and amplification of the cDNA in the presence of specific primers which followed by sequencing. The gene expression level of P5CS was done using real-time PCR methods. Thereafter the salts effects were analyzed on peroxidase and catalase activity and carotenoid, chlorophyll, total protein and proline contents were measured in the treated seedlings using spectrophotometer apparatus. Identification and characterization of P5CS mRNA were performed and its sequence partially registered in GeneBank. The data were shown that P5CS gene expression level and the proline content increased with increasing concentrations of NaCl up to 300 mM. Peroxidase and catalase activities as well as the total protein content were enhanced by increasing the concentrations of NaCl in all samples. Chlorophyll and carotenoid contents were significantly decreased by salinity stress. According to the results, a correlation was found between proline content and P5CS gene expression. Furthermore, these results also provide an insight into the response of this medicinal plant to salt stress.