Abstract: Quinclorac phytotoxicity to tobacco is one of the important problems affecting the tobacco material in China, and the phytotoxicity mechanism may be related to ethylene synthesis. In order to clarify the mechanism of quinclorac phytotoxicity, tobacco 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene family was identified, and its expression pattern under quinclorac phytotoxicity was analyzed. The results revealed that there were 19, 9 and 10 ACO genes in Nicotiana tabacum, Nicotiana sylvestris and Nicotiana tomentosiformis respectively. Subcellular localization analysis showed that almost all ACOs were localized in the cytoplasm. Phylogenetic analysis revealed that ACO family was classified into three clades with the most members in type Ⅰ and the least in type Ⅱ. Prediction of MicroRNA target sites found that six NtACO contained potential miRNA target sites. Cis-acting elements analysis showed that NtACO promoters contained various types of cis-acting elements involving hormone responsiveness, low-temperature responsiveness, defense and stress responsiveness, etc. Analysis of the expression pattern under quinclorac treatment indicated that all members of NtACO family were up-regulated by quinclorac, and Ntab0263610, Ntab0263620, Ntab0220530 and Ntab0220520 were most up-regulated, which suggested that these genes might play key roles in ethylene synthesis induced by quinclorac in tobacco.