Abstract: To elucidate the resistance of NtTIR1/AFBs family genes to auxin herbicides and the mechanism of quinclorac phytotoxicity in tobacco, the tobacco auxin receptor gene family was identified and their gene expression patterns were analyzed. The results showed that there were ten NtTIR1/AFBs genes in Nicotiana tabacum. Subcellular localization analysis showed that five NtTIR1/AFBs localize in the nucleus, three in the cytoplasm, and two in the chloroplasts. Phylogenetic analysis revealed that NtTIR1/AFBs were evolved into four classes, while AtTIR1/AFBs in Arabidopsis thaliana into three classes. Protein domain analysis revealed the presence of conserved F-box and LRR domains predominantly in NtTIR1/AFBs. Furthermore, there were differences in the number of domains between NtTIR1/AFBs and AtTIR1/AFBs. MicroRNA target analysis identified putative miRNA binding sites within four NtTIR1/AFBs genes, with 20083 and 20090 harboring eight miRNA target sites each. Tissue expression analysis revealed that the expressions of 20081, 20082, 20088 and 20091 were relatively high in all tissues. Notably, gene expression analysis after quinclorac treatment showed that the expressions of 20085 and 20086 were highly up-regulated in tobacco roots. 20082 had the highest up-regulated expression fold in tobacco leaves, while 20081 and 20090 had the highest down-regulated expression fold in tobacco leaves.