In this study, the oocysts of E. brunetti were isolated from chicken feces in Baoding city, Hebei province by single-oocyst isolation technique. The results of biol. characteristics indicated that the min. prepatent period was 125 h for the E. brunetti strain, with an average size of 26.1 μm×21.3 μm, and a shape index of 1.22. This parasite could cause hemorrhage of intestinal mucosa in rectum, cloaca, and the posterior part of small intestine. Infected with certain dose of this parasite, the performance trait of chickens were affected severely, and 2×105 per chicken oocysts could cause chickens to die, which indicated that the parasite had a relatively strong pathogenicity. The oocysts were further identified using specific PCR assays, and the results illustrated that the samples collected from single oocyst separation were mono-specific species of E. brunetti, which was named as BBD strain.