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Enterocytozoon hepatopenaei (EHP) is a newly emerged pathogen reported to retard the growth of cultured shrimp, including the tiger shrimp Penaeus monodon and the white shrimp Penaeus vannamei. Shrimp culture in Jiangsu Province in China has experienced explosive development in recent years, but this expansion has also increased the threat of retardation of shrimp growth. In this study, we investigated P. vannamei culture in earthen and greenhouse ponds in Jiangsu Province for EHP infection using light and transmission electron microscopy, histopathological analysis and polymerase chain reaction (PCR). Squash analysis of hepatopancreas showed a large number of EHP in heavily infected shrimp. Acidophilic inclusions and spore aggregations were observed in epithelial cells of hepatopancreas. Transmission electron microscopy revealed early- and late-stage plasmodia as multiple nuclei as well as mature spores measuring 1.1 μm × 0.7 μm, findings that are typical of the family Enterocytozoonndiae. A PCR product of ~ 779 bp was yielded and showed 97–99% identity with the EHP reported from Vietnam, Thailand, Latin America and India. PCR screening results showed that the prevalence of EHP in growth-retarded shrimp was relatively high (93%) in greenhouse ponds; this was approximately equal to that in growth-retarded shrimp in earthen ponds (91.3%). However, the prevalence of EHP in seemingly normal shrimp in greenhouse ponds (10.6%) was much lower than that in seemingly normal shrimp in earthen ponds (72.4%). This study showed that the prevalence of EHP in shrimp in Jiangsu Province was relatively high, whereas culturing shrimp in greenhouses appeared to limit the rate of EHP infection. [ABSTRACT FROM AUTHOR]