Blooms of the dinoflagellate Prorocentrum donghaiense occur frequently along the East China coast and Yangtze River estuary and pose a potential threat to coastal ecosystems. However, information on the genetic diversity of P. donghaiense populations in the East China Sea remains limited despite its ecological significance. Here we developed nine microsatellite markers based on transcriptome sequencing and analyzed genetic diversity and structure of P. donghaiense population in the East China Sea. We found that the Nei’s genetic diversity index of P. donghaiense population varied from 0.019 to 0.551 with an average of 0.227, whereas Shannon’s index ranged from 0.055 to 0.968 with an average of 0.431. Moreover, four genetically distinct subpopulations were identified based on STRUCTURE analysis. These results clearly indicated that P. donghaiense population during the blooms had high genetic diversity in the East China Sea, which might form the basis for variability in phenotype of P. donghaiense population and empower its capability to adapt to changing environment. Collectively our findings uncovered the genetic distinctiveness in P. donghaiense populations between 2014 and 2016 and provide valuable insights into the genetic structure and diversity of P. donghaiense blooms.