Objective It is aimed to investigate the prevalence and genotype distribution of Enterocytozoon bieneusi in experimental Beagle dogs in China.Method A total of 452 fresh fecal samples of experimental Beagle dogs were collected from Beijing， Nantong and Lianyungang during March to May 2025. Genomic DNA was extracted from the samples， and all samples were detected by nested PCR targeting the internal transcribed spacer （ITS） of E. bieneusi. Positive samples were further sequenced and analyzed.Result （1）The infection rates of E. bieneusi in experimental Beagle dogs in Beijing， Nantong and Lianyungang were 9.3%（14/150）， 14.8%（4/84） and 6.9% （15/218）， respectively， with an average infection rate of 7.3% （33/452）. （2）Sequence analysis identified four genotypes of E. bieneusi， including one known genotype PtEb IX and three novel genotypes （BGD-1， BGD-2， BGD-3）， among which PtEb IX was the dominant genotype in this study. （3）Phylogenetic tree analysis showed that all sequences obtained in this study were clustered with the dog-specific genotype PtEb IX， indicating that all genotypes identified in this study belonged to dog-specific genotypes.Conclusion E. bieneusi infection is confirmed in experimental Beagle dogs in China. To ensure the health of experimental animals and the reliability of scientific research， high attention should be paid to the prevention and control of E. cytozoon bieneusi in experimental Beagle dogs.