Valine-glutamine genes (VQ) acted as transcription regulators and played the important roles in plant growth and development, and stress tolerance through interacting with transcription factors and other co-regulators. In this study, sixty-oneVQgenes containing the FxxxVQxxTG motif were identified and updated in theNicotiana tobacumgenome. Phylogenetic analysis indicated thatNtVQgenes were divided into seven groups and genes of each group had highly conserved exon-intron structure. Expression patterns analysis firstly showed thatNtVQgenes expressed individually in different tobacco tissues including mixed-trichome (mT), glandular-trichome (gT), and nonglandular-trichome (nT), and the expression levels were also distinguishing in response to methyl jasmonate (MeJA), salicylic acid (SA), gibberellic acid (GA), ethylene (ETH), high salinity and PEG stresses. Besides, onlyNtVQ17of its gene family was verified to have acquired autoactivating activity. This work will not only lead a foundation on revealing the functions ofNtVQgenes in tobacco trichomes but also provided references toVQgenes related stress tolerance research in more crops.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40529-023-00376-x.