Giardia duodenalis is a significant zoonotic parasite. In this study, 767 fresh fecal samples were collected randomly from six large-scale sheep farms in Southern Xinjiang, China. Initially, G. duodenalis was screened using PCR at the SSU rRNA gene. Positive samples then underwent PCR amplification at the bg, gdh, and tpi genes. The prevalence of G. duodenalis in sheep was 17.5% (134/767), with the highest prevalence observed in the 3-6 months age group at 26.8% (56/209) and the lowest in the over 12 months age group at 6.8% (14/205). Among the 134 positive samples, only Assemblage E was identified. A total of 106, 92, and 98 sequences of G. duodenalis were obtained at the gdh, tpi, and bg genes, respectively. Fourteen isolates of G. duodenalis were successfully amplified at all three genes, resulting in nine G. duodenalis multilocus genotypes (MLG) named MLG E1-MLG E14, indicating high genetic diversity. In conclusion, G. duodenalis infection in sheep from large-scale farms is common in Southern Xinjiang, China, showing geographical regional distributions and genetic diversity.