Fifty-eight Giardia duodenalis-positive fecal specimens from three dairy farms in Georgia, USA were genotyped and subtyped by sequence analysis of the triosephosphate isomerase gene. Both the livestock-specific assemblage E and the potentially zoonotic assemblage A were found, with the former assemblage detected in 83% of the specimens. A high degree of genetic polymorphism was evident within assemblage E, with 11 distinct subtypes identified, eight of which represented new subtypes. Three subtypes were identified in assemblage A, with the subtype A2 transiently found in calves and cows on one farm. All farms had multiple assemblage E subtypes circulating in cattle at each sampling, and concurrent infection with mixed subtypes or genotypes occurred in 24% of animals. Thus, the high intensity of G. duodenalis transmission is not only reflected by the high prevalence of the infection but also by the high intragenotypic diversity and concurrent occurrence of mixed infections. The zoonotic potential of bovine G. duodenalis needs to be further studied by extensive characterization of assemblage A specimens at the subtype level.