Simple Summary: Giardia duodenalis is a common zoonotic intestinal protozoan and causes significant economic losses and potential risks to public health. China is among the several major producers of donkeys worldwide, with approximately 2.6 million donkeys being reared in 2019, but studies on G. duodenalis infection in donkeys were limited. Thus, in order to better understand the molecular epidemiology of G. duodenalis in donkeys in China, 815 fecal samples were collected from donkeys in three representative geographical locations in Shanxi Province, North China, to determine the presence and genotypes of G. duodenalis based on three established loci (tpi, bg, and gdh). A total of 137 out of 815 fecal samples were detected as G. duodenalis-positive with an overall prevalence of 16.81%. Among the detected assemblages A, B, and E, the most predominant assemblage was B in the three study areas. Three MLGs (MLG-novel-1 to 3) were identified with multilocus genotypes (MLGs) analysis and validated by a phylogenetic tree. This is the first report of G. duodenalis infection in donkeys in Shanxi Province, which not only enhances our understanding of the genetic diversity of G. duodenalis in donkeys in China but also provides essential baseline data for the prevention and control of G. duodenalis infection donkeys in the study areas. Giardia duodenalis is a ubiquitous flagellated protozoan, causing significant economic losses to animal husbandry and posing threats to public health. China ranks the world's sixth largest major producer of donkeys, rearing approximately 2.6 million donkeys in 2019, but limited investigation of G. duodenalis prevalence has been conducted in the past, and it is yet to be known whether donkeys in Shanxi Province are infected with G. duodenalis. In the present study, a total of 815 fecal samples collected from donkeys in representative regions of Shanxi Province, North China, were examined for G. duodenalis using nested PCR. Then, the assemblages and multilocus genotypes (MLGs) were examined based on three established loci: namely, β-giardin (bg), triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh). The overall prevalence of G. duodenalis in donkeys in Shanxi Province was 16.81% (137/815). The region was identified as the main risk factor for the observed difference in G. duodenalis prevalence in donkeys among the three study areas (χ2 = 21.611, p < 0.001). Assemblages A, E, and B were identified, with the latter as the predominant assemblage. Three MLGs (MLG-novel-1 to 3) were formed based on sequence variation among the three loci. The present study reveals the presence of G. duodenalis in donkeys in Shanxi Province, North China, for the first time, which not only enriches the data on the distribution of G. duodenalis in donkeys in China but also provides useful baseline data for planning control strategies against G. duodenalis infection in the sampled areas. [ABSTRACT FROM AUTHOR]