This research explored the occurrence and molecular diversity of Enterocytozoon bieneusi in sheep and goats across Jiangsu Province. A total of 786 fresh fecal specimens were gathered from 18 farms in five regions and analyzed by nested PCR amplification and sequencing of the ribosomal internal transcribed spacer (ITS). E. bieneusi DNA was detected in all surveyed areas, with infection levels varying from 23.65% to 42.81%, resulting in an overall positivity rate of 36.51% (287/786). No significant statistical differences were observed between sheep and goats or among different age classes (p > 0.05), but infection rates differed notably according to the animals’ health status (p < 0.05). Sequence and cloning results identified six distinct genotypes—BEB6, CHG2, CHG3, CHC8, CHG14, and COS-I—all belonging to Group 2, a cluster previously regarded as non-zoonotic.
