Core primers are important for genetic diversity analysis, germplasm identification, purity and genuineness test, and fingerprint establishment. Currently, we have screened 20 tobacco germplasm stocks with distant relationships by using of 278 SSR primer pairs, which evenly distribute on 24 chromosomes, through the strategy of distant relationships, and among which 32 primer pairs were selected out. And then, another 10 germplasm stocks with similar relationship were used to screen the 32 primer pairs, and 14 SSR core primer pairs were obtained. These 14 SSR core primer pairs were conducted on pedigree research, genetic diversity analysis and agronomic traits clustering study for 39 germplasm stocks. The result showed these core primer pairs could be applied to germplasm identification and genetic diversity study in tobacco.