BackgroundGlutathione S-transferases (GSTs) are large and multifunctional proteases that play an important role in detoxification, protection against biotic and abiotic stresses, and secondary metabolite transportation which is essential for plant growth and development. However, there is limited research on the identification and function ofNtGSTs.ResultsThis study uses K326 and other six tobacco varieties (Hongda, HG, GDH11, Va116, VG, and GDH88) as materials to conduct comprehensive genome-wide identification and functional characterization of theGSTgene in tobacco. A total of 59NtGSTs were identified and classified into seven subfamilies via the whole-genome sequence analysis, with the Tau type serving as the major subfamily. TheNtGSTs in the same branch of the evolutionary tree had similar exon/intron structure and motif constitution. There were more than 42 collinear blocks between tobacco and pepper, tomato, and potato, indicating high homology conservation between them. Twelve segmental duplicated gene pairs and one tandem duplication may have had a substantial impact on the evolution and expansion of the tobaccoGSTgene family. The RT-qPCR results showed that the expression patterns ofNtGSTs varied significantly among tissues, varieties, and multiple abiotic stresses, suggesting thatNtGSTgenes may widely respond to various abiotic stresses and hormones in tobacco, includingNtGSTF4,NtGSTL1,NtGSTZ1, andNtGSTU40.ConclusionsThis study provides a comprehensive analysis of theNtGSTgene family, including structures and functions. ManyNtGSTsplay a critical regulatory role in tobacco growth and development, and responses to abiotic stresses. These findings offer novel and valuable insights for understanding the biological function ofNtGSTsand the reference materials for cultivating highly resistant varieties and enhancing the yield and quality of crops.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09450-x.