The prevalence of Giardia duodenalis genotypes was determined in adult dairy cows. Fecal specimens were collected from two farms each in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens, cleaned of fecal debris and concentrated using CsCl density gradient centrifugation, were subjected to PCR and DNA sequence analysis. The prevalence of G. duodenalis infection, ranged from 3% to 64%, with an average prevalence of 27% (144 positive cows out of 541 examined). DNA sequence analysis of the 16S rRNA gene revealed the presence of both Assemblage A and Assemblage E, G. duodenalis. Overall, Assemblage E was present in 25% of all animals tested and Assemblage A was present in 2% of the animals. As a percentage of G. duodenalis isolates, Assemblage E represented 94% and Assemblage A represented 6%. Although, most of the cows were infected with a genotype that is not known to be infectious for humans, adult cows on five farms did harbor varying levels of zoonotic Assemblage A, G. duodenalis. Therefore, although adult cows do not appear to be a significant source of human infectious cysts in the environment, the risk from this age group should not entirely be discounted.