Technical Abstract: To determine the prevalence of Giardia duodenalis in weaned beef calves in cow-calf operations in the United States, fecal specimens were collected from 819 calves (6 to18 months of age) in 20 states from 49 locations. After cleaning and concentration procedures to maximize the potential recovery of cysts from feces, DNA was extracted from each of the 819 specimens. The presence of G. duodenalis was determined by nested PCR of a fragment of the SSU rRNA gene. All positive PCR products were subjected to sequence analysis. The overall prevalence of Giardia was 33.5 % with prevalence ranging from 0 to 100% among operations. The highest prevalence of infected beef calves was found in cow-calf operations from Georgia, Idaho, Nebraska, and Iowa with 100, 90, 87.5, and 85% of the animals infected, respectively. Giardia was not detected in 7 operations, 2 cow-calf operations each from Louisiana and Oklahoma, and 1 each from Texas, South Dakota, and California. The molecular analysis of the 274 Giardia-positive samples identified Assemblage E in 260 (31.7%) and Assemblage A in 10 (1.2%). In four calves from a farm in Nebraska a mixed infection with Assemblage A and E was observed. The potentially zoonotic assemblage A was detected in specimens from four operations in Nebraska, and one in each in Iowa and Oregon. These findings indicate that most G. duodenalis found in beef cattle was Assemblage E which represents no known zoonotic threat. However, the presence of Assemblage A in a small number of animals poses a poses a potential risk of infection to humans.