A total of 791 microsatellites (SSRs) were isolated from 7055 Panax ginseng expressed sequence tags (ESTs). According to primer design criteria, 68 primer pairs for EST-SSR were designed. Under an appropriate polymerase chain reaction (PCR) system, all EST-SSR primer pairs were screened against genomic DNA of Ji'anchangbo and Fusong'ermaya from Panax ginseng, and 43 EST-SSR primer pairs out of the above 68 resulted in PCR products. Then, all 43 pairs were detected in nine P. ginseng, two Panax quinquefolius and two Acanthopanax senticosus cultivars for polymorphisms, and 26 pairs (60.47%) were found to be polymorphic, accounting for 38.23% of the total number of designed primer pairs. These results demonstrate the possibility of developing EST-SSR markers using P. ginseng ESTs.