BACKGROUND Giardia duodenalis is an important intestinal parasitic protozoan that infects several vertebrates, including humans. Cattle are considered the major source of giardiasis outbreak in humans. This study aimed to investigate the prevalence and multilocus genotype (MLG) of G. duodenalis in Shanxi, and lay the foundation for the prevention and control of Giardiosis. RESULTS DNA extraction, nested polymerase chain reaction, sequence analysis, MLG analysis, and statistical analysis were performed using 858 bovine fecal samples from Shanxi based on three gene loci: β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi). The overall prevalence of G. duodenalis was 28.3%, while its prevalence in Yingxian and Lingqiu was 28.1% and 28.5%, respectively. The overall prevalence of G. duodenalis in dairy cattle and beef cattle was 28.0% and 28.5%, respectively. G. duodenalis infection was detected in all age groups evaluated in this study. The overall prevalence of G. duodenalis in diarrhea and nondiarrhea samples was 32.4% and 27.5%, respectively, whereas that in intensively farmed and free-range cattle was 35.0% and 19.9%, respectively. We obtained 83, 53, and 59 sequences of bg, gdh, and tpi in G. duodenalis, respectively. Moreover, assemblage A (n = 2) and assemblage E (n = 81) by bg, assemblage A (n = 1) and assemblage E (n = 52) by gdh, and assemblage A (n = 2) and assemblage E (n = 57) by tpi were identified. Multilocus genotyping yielded 29 assemblage E MLGs, which formed 10 subgroups. CONCLUSIONS To the best of our knowledge, this is the first study to report cattle infected with G. duodenalis in Shanxi, China. Livestock-specific G. duodenalis assemblage E was the dominant assemblage genotype, and zoonotic sub-assemblage AI was also detected in this region.