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Blastocystis is a highly prevalent eukaryotic parasite of many animals and humans worldwide. It can compromise the gastrointestinal tract and cause gastrointestinal symptoms, constituting a serious threat to human health and animal growth. Many animals are potential sources of Blastocystis infection in humans. However, limited data are available regarding the prevalence and subtype distribution of Blastocystis infection among zoo animals in China. Therefore, the present study examined the prevalence and subtypes of Blastocystis in zoo animals in Hangzhou, Dalian, and Suzhou cities, China. Of 450 fecal samples from zoo animals, 27 (6.0%) were PCR-positive for Blastocystis, with 7.7% (8/104), 11.3% (7/62), 16.7% (3/18), 1.8% (2/114), 6.3% (1/16), 9.5% (2/21), and 3.6% (4/109) in artiodactyla, aves, rodentia, nonhuman primates, perissodactyla, marsupialia, and carnivora, respectively. Significant differences in the prevalence of Blastocystis were found among different animal groups (P < 0.05). Sequence analysis showed 7 known subtypes (ST2, ST4, ST5, ST7, ST8, ST10, and ST14) of Blastocystis in the present study, with ST10 (10/27) as the predominant subtype in all three of the examined zoos. To our knowledge, this is the first report of Blastocystis infection in Damaliscus dorcas, Cervus elaphus, Macropus rufogriseus, Grus japonensis, Trichoglossus haematodus, Panthera tigris ssp. tigris (white), Panthera tigris ssp. altaica, Lycaon pictus, Suricata suricatta, and Dolichotis patagonum in China. These results demonstrate the presence of Blastocystis infection in zoo animals and provided baseline data for preventing and controlling Blastocystis infection in zoo animals and humans in China. [ABSTRACT FROM AUTHOR]