In order to explore the role of NBSLRRlike gene RPS2 in the process of resistance to leaf stripe disease in hulless barley, we took leaf stripe disease resistant variety Kunlun 14 and susceptible variety Z1141 as materials in study. HvnRPS2 gene sequence by designing primer cloning according to transcriptome sequence (RNAseq), and carried out a series of bioinformatics analysis, we used qRTPCR to analyze the expression pattern of HvnRPS2 gene in different resistant hulless barley varieties. The result showed: (1) its fulllength sequence of 3 089 bp, without introns, contained a 2 760 bp open reading frame, encoded 919 amino acids, had a theoretical isoelectric point of 5.93, and had a predicted protein molecular weight of 104.2 kDa. The protein encoded by HvnRPS2 is hydrophilic and its secondary structure is mainly composed of irregular curl and αhelix. (2) The protein multiple sequence alignment and phylogenetic tree analysis showed, HvnRPS2 has highly conserved NBARC and LRR domains, and belonged to the NBSLRR gene family. and the closest relationship with HvRPS2 in Hordeum vulgare and OsRPS2 in Oryza. (3) qRTPCR results showed that HvnRPS2 gene decreased first, then increased and finally decreased with the prolongation of leaf stripe stress. Compared with normal leaves, the expression level of HvnRPS2 gene in susceptible leaves was significantly downregulated, and the downregulation value of susceptible varieties was significantly higher than that of disease resistant varieties (P < 0.01). The research speculates that HvnRPS2 plays an important negative role in the resistance of hulless barley to leaf stripe disease. The results of the study lay the foundation for further exploring the regulation mechanism of this gene in the resistance of barley to leaf stripe disease.