The aim of this study was to construct a DNA fingerprinting database of 52 sweetpotato germplasm from ‘The National Germplasm Guangzhou Sweetpotato Nursery Genebank’ using EST-SSR. Sixteen EST-SSR core primer pairs with high polymorphisms and good repeatability were successfully screened out from 324 candidates. The 16 core primer pairs amplified a total of 159 alleles among the 52 sweetpotato germplasms. Alleles amplified by each pair of primers ranged from 5 to 19, with a mean of 9.94. The polymorphic information content values (PIC) ranged from 0.6235 to 0.9593, with a mean of 0.7991. Two primers were selected to construct the fingerprinting database. Clustering analysis by the software of NTSYS indicated that EST-SSR markers could effectively detect the genetic relationships among the various sweetpotato gersmplasms.