The composition and structure of gut microbiota are easily influenced by external factors, especially host genetics. While horses are closely related to human life, there is a lack of research comparing the gut microbiota compositions and differences between native Mongolian horse (MH) and imported Dutch Warmblood horse (DH). Here, we collected feces from MH and DH and compared the differences in gut microbiota between the two breeds of horses using amplicon sequencing. Results showed that there was no significant difference in the diversity of gut microbiota between the two breeds of horses. At the phylum level, both Firmicutes and Bacteroidota were the most dominant phyla in all samples, independent of species. In addition, we also observed significant differences in 78 bacterial genera between the MH and DH, of which 36 genera (Bifidobacterium, Chujaibacter, Lactobacillus, Rothia, etc.) were significantly increased in DH and 42 genera (Aequorivita, Aeromicrobium, Psychrobacillus, Brevibacterium, etc.) were significantly decreased compared with MH. Altogether, this investigation dissected the compositions and differences of the gut microbiota between DH and MH and observed distinct differences in the gut microbiota between the two breeds of horses. Furthermore, these findings enhance our comprehension of the gut microbiome features of distinct horse breeds, which helps to analyze the differences in traits among different horses.