Giardia duodenalis is an important zoonotic intestinal parasite responsible for diarrhea in humans and other animals worldwide. The present study was conducted to assess the prevalence of bovine giardiosis and to perform molecular characterization of Giardia duodenalis in the northeast of China. A total of 655 fecal specimens were collected from dairy cattle in 15 farms located in three different provinces. G. duodenalis assemblages and subtypes were determined by sequence analysis of the triosephosphate isomerase (TPI) gene. As a whole, the G. duodenalis infection rate in dairy cattle was 7.9% (52/655), as determined by Lugol's iodine staining. Two assemblages were identified, namely, the potentially zoonotic assemblage A (n = 1), the livestock-specific assemblage E (n = 50), and a mixed infection case of assemblages A and E. Seven distinct subtypes of E assemblages were identified and E-XI, E-I and E-III are the major subtypes. Only subtype A-I was identified in assemblage A. Findings relevant to assemblage A are of public health importance. The results indicated the livestock-specific assemblage E is the major genotype and zoonotic assemblage A or B occurs very seldomly which is significantly different with previous report in the same area. So that determination of genotypes in individual epidemiological setting can make important contributions to public health.