Giardia duodenalis is a gastrointestinal parasitic protozoan with important veterinary and public health implications worldwide. In this study, a total of 1440 fecal specimens were collected from dairy calves in Guangdong Province, China. The overall prevalence of G. duodenalis was 2.2% (31/1440) by polymerase chain reaction (PCR). The prevalence was 8.4% and 1.1% in pre- and post-weaned calves, respectively. Both assemblages A (6.5%, 2/31) and E (93.5%, 29/31) were found based on sequence analysis of the SSU rRNA gene. All G. duodenalis-positive samples were assayed with PCR followed by sequencing the beta giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes, and 20, 23, and 18 sequences were obtained, respectively. Multilocus genotyping yielded 10 assemblage E multilocus genotypes (MLGs). All assemblage E MLGs were genetically distinct from those obtained in cattle from Henan, Xinjiang, Shanghai, and Shaanxi in China. This finding was also supported by FST, Nm, and median-joining network analysis. Results of this study indicated that G. duodenalis was found in low prevalence in dairy calves in Guangdong Province, southern China; nevertheless, the infected calves might represent a public health risk in this region. Additionally, the differences in assemblage E MLGs from the five analyzed regions revealed significant geographic segregation of G. duodenalis in China.