Abstract: Chalcone synthase (CHS) catalyzes the first committed step for the biosynthesis of flavonoids, which are important for tobacco quality. To extend the knowledge on tobacco CHSs, we conducted comparative analyses of CHS genes among allotetraploid tobacco and its diploid progenitors. In this study, 8, 4, and 4 CHS genes were identified from N. tabacum, N. sylvestris and N. tomentosiformis, respectively. All the tobacco CHS genes contained one intron, and shared two highly conserved domains, Chal_Sti_Synt_N and ACP_Syn_Ⅲ_C. The putative polypeptides of tobacco CHSs contained 389 to 420 amino acids with predicted molecular weights ranging from 40.74 to 46.02 kDa. Sequence and phylogenetic analyses revealed that each NtCHS gene in the allotetraploid tobacco had one ortholog in both diploid genomes. The chromosome location analysis of NtCHSs suggested that CHSG and CHSJ genes were deduced to be closely linked with each other in tobacco genomes. The spatial expression analysis in three tobacco species showed the expression of all tobacco CHS genes exhibited lower levels in stems than those in leaves and roots, and some NtCHS homologous pair members also showed distinct expression patterns. The reproductive tissues had the highest accumulation of mRNA transcripts of NtCHS genes. In addition, most NtCHS genes were actively involved in the process of tobacco response to ABA, GA, MeJA, and many abiotic stresses including drought, salinity, darkness and phosphate starvation. This study provides new information about tobacco CHSs by revealing their real functions in tobacco flavonoids biosynthesis, as well as their resistance to abiotic stresses.