Abstract: Cytochrome P450 (CYP450) is a supergene family which encodes multifunctional oxidase, playing an important role in biosynthesis, metabolic detoxification, and plant defense. In CYP450 family, the CYP71D subfamily genes mainly function in secondary metabolite synthesis and pest control. In order to better understand the genetic characteristics and functions of tobacco CYP71D subfamily genes, their gene structure and expression pattern were analyzed by bioinformatics tools and transcriptome data. The results showed that there are 42 members in tobacco CYP71D subfamily and they were distributed on the chromosome unevenly. Besides, their amino acid lengths and isoelectric points were quite different, while their gene structure and conserved-domain distribution were highly consistent. The secondary structure analysis showed that CYP71D subfamily members contained the typical conserved domains in P450 proteins. The gene expression pattern analysis showed that most CYP71D genes were specifically expressed in roots, leaves and flowers, and some of them were induced by various elicitors, including phytohormone IAA, temperature and black shank. This study contributes to further researches in the function of CYP71D subfamily genes and breeding for stress-resistant tobacco cultivars.