Ulva species can grow rapidly in nutrient-rich habitats causing green tides and marine fouling. A more complete understanding of the reasons behind these outbreaks is urgently required. Accordingly, this study attempts to use microsatellite markers based expressed sequence tag(EST) to analyze the genetic variation of several Ulva prolifera populations in the South Yellow Sea of China. Two hundred and thirty-eight SSRs were identified from 8 179 unique ESTs(6 203 newly sequenced and 1 976 downloaded from NCBI database) and 37 primer pairs were successfully designed according to the ESTs; 11 pairs were selected to detect the genetic diversity and relationship of 69 attached U. prolifera samples and 13 free-floating samples collected from coastal and off-coast areas of the South Yellow Sea. The results of cross-species transferability showed that six of the 11 EST-SSR primers could give good amplification in other five Ulva species and the average allele number was 4.67. Genetic variation analysis indicated that all 82 U. prolifera samples were clearly divided and most samples collected from the same site clustered together as a group in the dendrogram tree produced by unweighted pair-group mean analysis(UPGMA) method and the cluster results showed some consistency with the geographical origins. In addition, 13 free-floating samples(except HT-001-2) were grouped as a single clade separated from the attached samples. Ulva species can grow rapidly in nutrient-rich habitats causing green tides and marine fouling. A more complete understanding of the reasons behind these outbreaks is urgently required. genetic variation of several Ulva prolifera populations in the South Yellow Sea of ​​China. Two hundred and thirty-eight SSRs were identified from 8 179 unique ESTs (6 203 newly sequenced and 1 976 downloaded from NCBI database) and 37 primer pairs were successfully designed according to to the ESTs; 11 pairs were selected to detect the genetic diversity and relationship of 69 attached U. prolifera samples and 13 free-floating samples collected from coastal and off-coast areas of the South Yellow Sea. The results of cross-species transferability showed that six of the 11 EST-SSR primers could give good amplification in other five Ulva species and the average allele number was 4.67. Genetic variation analysis indicated that all 82 U. prolifera samples were clearly divided and most samples collected from the same site clustered together as a group in the dendrogram tree produced by unweighted pair-group mean analysis (UPGMA) method and the cluster results showed some consistency with the In addition, 13 free-floating samples (except HT-001-2) were grouped as a single clade separated from the attached samples.