To determine the prevalence of bovine giardiasis in Heilongjiang Province in China and to molecularly characterize Giardia duodenalis, feces were collected from 814 dairy and beef cattle ranging in age from 6 days to 9 years. Clinical symptoms of diarrhea were recorded at the time of sampling. The G. duodenalis infection rate in cattle was 5.2 % (42/814) as determined by Lugol's iodine staining. G. duodenalis assemblages and subtypes were genetically diagnosed by sequence analysis of the triosephosphate isomerase (TPI) gene. Three assemblages were identified, representing A (n = 1), B (n = 18), and E (n = 24), with a mixed infection case of assemblages A and E. High heterogeneity was also observed within assemblages B and E at the TPI locus. Among the assemblages, eight subtypes of assemblage B and three subtypes of assemblage E were found to be novel subtypes. Findings on assemblages A and B are of public health importance. The zoonotic potential of bovine giardiasis needs to be further assessed by extensive genetic data of assemblages A and B from humans at the subtype level. The newly found subtypes of assemblages B and E imply that the evaluation of geographically distributed subtypes is of importance.