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A total of 500 fecal samples were collected from Equus animals in six different cities (Ardabil, Namin, Nir, Meshginshahr, Germi, and Khalkhal) of Ardabil Province, northwestern Iran, with 200 samples from horses, 200 from donkeys, and 100 from mules. Of the horse samples, 100 were from racing horses under special monitoring and care, while the remaining 100 were from non-racing horses, including those used for herding or in rural areas. All fecal samples were examined for the presence of Blastocystis sp. using PCR amplification of the SSU rRNA gene's barcode region after DNA extraction. The molecular prevalence of Blastocystis infection in Equus animals was 7.6% (38/500). Blastocystis was more common in horses [11.5% (23/200)] than in donkeys [5.5% (11/200)] and mules [4% (4/100)] (P > 0.05). Compared to racing horses [3% (3/100)], non-racing/rural horses [20% (20/100)] exhibited a substantially higher prevalence of Blastocystis (P < 0.05). The prevalence of Blastocystis in diarrheal samples and younger animals was remarkably higher than in formed samples and older animals, respectively (P < 0.05). No significant difference in Blastocystis infection prevalence was found between the genders of examined animals (P > 0.05). In Equus animals, 38 Blastocystis isolates included eight STs: ST10 [31.6% (12/38)], ST1 [21.1% (8/38)], ST2 [15.8% (6/38)], ST3 [10.5% (4/38)], ST4 [7.9% (3/38)], ST7 [5.2% (2/38)], ST14 [5.2% (2/38)], and ST6 [2.6% (1/38)]. These results suggest that Equus animals act as a proper reservoir for numerous Blastocystis STs, consequently playing a crucial part in the spread of this protozoan infection to humans, animals, and water reservoirs. • The present study is the first large-scale research conducted in Iran with the aim of determining the prevalence, STs distribution, and zoonotic importance of Blastocystis in Equus animals (horses, donkeys and mules). • The molecular prevalence of Blastocystis infection in Equus animals was 7.6% (38/500). • Blastocystis was more common in horses [11.5% (23/200)] than in donkeys [5.5% (11/200)] and mules [4% (4/100)]. • Compared to racing horses [3% (3/100)], non-racing/rural horses [20% (20/100)] exhibited a substantially higher prevalence of Blastocystis (P < 0.05). • All STs (ST1-ST4, ST6, ST7, ST10, and ST14) isolated from Equus animals in this study are in the group of zoonotic STs. [ABSTRACT FROM AUTHOR]