The invention relates to a molecular marker Nicotine Associated SNP 1 and kit for identifying nicotine content of tobacco as well as application thereof, and belongs to the technical field of molecular biology. The molecular marker Nicotine Associated SNP 1 is an SNP site Nitab4.5_0002539: 95304 A/G which is positioned at the 95304 site of a genome segment No.0002539 of a tobacco genome version Nitabv4.5Genome Scaffolds Edwards2017. Tobacco germplasm resources with different nicotine content can be accurately identified and screened by 100%, and the screened tobacco material can be directly used for breeding new tobacco varieties without a transgenic method.>