Thirteen NtPPO genes were identified from the newly published genome of Nicotiana tabacum L. using bioinformatics methods. The gene structures of the family members were similar, and their protein motifs were conserved. All NtPPOs were mapped to specific chromosomes. The results of phylogenetic analysis showed that tobacco polyphenol oxidase(PPO) proteins are more closely related to those of tomato and potato, but are distantly related to those of soybean. The middle and upper mature leaves of the cultivars‘CB-1’and‘K326’were used as materials. Photoshop pixel method and qRT-PCR were used to analyze the browning characteristics,PPO activity and expression of PPO family genes in tobacco leaves at the later yellowing and color-fixing stages. The results showed that during the curing process, the browning rate and PPO activity of tobacco leaves of cultivars‘CB-1’and‘K326’changed synchronously, and exhibited a highly significant positive correlation. The browning rate and PPO activity of cultivar‘CB-1’were significantly higher than those of cultivar‘K326’. The expression patterns of NtPPO genes varied with leaf position, cultivar, and curing stage. The expression levels of most NtPPO genes in the upper leaves were higher than those in the middle leaves, while NtPPO11 and NtPPO13 were not expressed in either cultivar. The expression levels of most NtPPO genes in‘CB-1’leaves were higher than those in‘K326’ leaves at the later yellowing and color-fixing stages, which was basically consistent with the trend of PPO activity. In summary, during the curing process of tobacco leaves, differential expression of PPO family genes affects PPO activity, thereby leading to the differences in the degree of browning of tobacco leaves.