Abstract: To rapidly screen out the primers suitable for the molecular biology of sugar beet from EST-SSR primers of sugar beet, 12 varieties of sugar beet were used to amplify with 80 pairs of EST-SSR primers of sugar beet. At the same time, 12 kinds of annealing temperatures were set and 8% non-denaturing PAGE was used to separate the PCR products. The results showed that although all 80 pairs of EST-SSR primers could amplify products, most of them were useless because they were not polymorphic or difficult to distinguish. There were only 13 pairs of primers amplifying bands which were clear and easy to distinguish. Among them, 4 pairs had high polymorphism and the other 9 pairs had low polymorphism. While the gradient annealing temperature indicated that the annealing temperature had no obvious influence on EST-SSR primers and only several primers produced unspecific bands under low annealing temperature. So the annealing temperature of all primers was set to be 60 ℃. Quick screening of EST-SSR primers could be achieved using different varieties and gradient annealing temperature. But more EST-SSR primers should be screened to get primers with high polymorphism.