Abstract: The SHI-related sequence(SRS) gene family encodes a class of plant-specific zinc finger protein transcription factors that play crucial roles in regulating plant growth, development and stress responses. To explore the function of the SRS gene family in tobacco, this study identified 18 NtSRS genes from the tobacco genome database, designated as NtSRS1 to NtSRS18. Bioinformatics analyses were conducted to investigate their phylogenetic relationships, gene structures, protein characteristics, subcellular localization, and promoter elements. Additionally, expression patterns of NtSRS family across different tissues and organs, as well as their responses to drought, salt, and low temperature stress, were analyzed using qRT-PCR. Results revealed that NtSRS proteins contain the conserved DUF702 domain, with most localized in the nucleus. Phylogenetic analysis clustered the NtSRS family into 5 subfamilies, with members of the same subfamily exhibiting highly conserved gene and protein structures. Promoter analysis identified numerous hormone-responsive elements and stress-responsive elements within the regulatory regions of NtSRS genes. Expression profiling showed that NtSRS genes exhibit significant tissue-specific expression, with most highly expressed in flower buds and stems. Notably, NtSRS3 and NtSRS16 were strongly induced under drought, salt, and low temperature stress conditions. Overall, this study provides a comprehensive characterization of the tobacco SRS gene family, offering a theoretical basis for the genetic improvement of tobacco germplasm resources.