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<i>Giardia duodenalis</i> 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 <i>G. duodenalis</i> prevalence has been conducted in the past, and it is yet to be known whether donkeys in Shanxi Province are infected with <i>G. duodenalis</i>. In the present study, a total of 815 fecal samples collected from donkeys in representative regions of Shanxi Province, North China, were examined for <i>G. duodenalis</i> using nested PCR. Then, the assemblages and multilocus genotypes (MLGs) were examined based on three established loci: namely, β-giardin (<i>bg</i>), triosephosphate isomerase (<i>tpi</i>), and glutamate dehydrogenase (<i>gdh</i>). The overall prevalence of <i>G. duodenalis</i> in donkeys in Shanxi Province was 16.81% (137/815). The region was identified as the main risk factor for the observed difference in <i>G. duodenalis</i> prevalence in donkeys among the three study areas (χ² = 21.611, <i>p</i> < 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 <i>G. duodenalis</i> in donkeys in Shanxi Province, North China, for the first time, which not only enriches the data on the distribution of <i>G. duodenalis</i> in donkeys in China but also provides useful baseline data for planning control strategies against <i>G. duodenalis</i> infection in the sampled areas.