Enterocytozoon bieneusi is an obligate intracellular parasitic fungi that infects a wide range of mammalian hosts. However, the literature is lacking information regarding the presence and diversity of E. bieneusi genotypes in domesticated dogs in Northwestern China. Fecal samples from 604 pet dogs were obtained in 5 cities (Urumqi, Korla, Hotan, Aksu, and Shihezi) in Xinjiang. Screening for E. bieneusi was performed, and isolates were genotyped via nested-PCR amplification of the internal transcribed spacer (ITS) of nuclear ribosomal DNA. The infection rate of E. bieneusi was 6.3% (38/604). The prevalence of E. bieneusi infections in adult animals (>1 year, 10.3%, 15/145) was higher than that in younger (≤1 year) dogs (5.0%, 23/459), which was statistically significant (p = 0.021). No significant difference was observed between the different collection sites or between sexes. Eight distinct genotypes were identified, including 5 known genotypes (PtEb IX, EbpC, D, CD9, and Type IV) and 3 novel genotypes (CD11, CD12, CD13). The most prevalent was genotype PtEb IX, being observed in 50.0% (19/38) of the samples, followed by EbpC (31.6%, 12/38), D (5.3%, 2/38), and the remaining genotypes (CD9, Type IV, CD11, CD12, and CD13) were observed in 1 sample (2.6%, 1/38) each. These findings suggest that genotypes PtEb IX and CD9 are canine host-adapted, and likely pose little risk of zoonotic transmission. Moreover, known zoonotic genotypes EbpC, D, and Type IV represent a public health concern and should undergo further molecular epidemiological investigation.