Purpose <p>Enterocytozoon bieneusi (E. bieneusi) is a microsporidium that causes intestinal disease in animals and humans, and transmitsperson to person and/or animals to people. A survey was conducted to determine the prevalence and genetic diversity E. bieneusiinfection in cattle in Hebei Province, China.</p> Methods <p>A total of 2,746 cattle fecal samples were collected from 11 cities in Hebei Province. PCR amplification targeting the ITS gene of E.bieneusi was performed to detect infections. The subtypes, sequence signatures, pairwise genetic distances, and genetic diversityindices were analyzed using MEGA, PhyloSuite, and PopART.</p> Results <p>The overall prevalence of E. bieneusi in cattle was 4.70% (129/2746). The infection rate varied significantly by region, age, breedingmode, and cattle type: higher in southern Hebei, cattle under one year old, intensively raised cattle, and dairy cattle. Six genotypeswere identified (CHG3, SX1, BEB4, BEB6, BEB8, and J), with BEB6 being the dominant genotype. Sequence analysis revealed 260polymorphic and conserved sites, including 41 variable sites (15.77% of total nucleotides). The nucleotide diversity index (Pi) was0.06472, and the haplotype diversity index (Hd) was 1.000.</p> Conclusion <p>This study provides the first comprehensive epidemiological data on E. bieneusi infection in cattle in Hebei Province and highlights itsrich genetic diversity. These findings offer insights into the potential risk of zoonotic transmission and serve as a reference for furthercontrol and prevention strategies.</p>