The flagellate parasite Giardia duodenalis is a major cause of diarrhoea in humans and in animals worldwide. Molecular techniques are particularly useful for studying the taxonomy, the population structure, the zoonotic potential of animal isolates, and the correlation between the genetic variability of the parasite and the range of clinical symptoms observed in humans. In this work, a new PCR assay that targets the beta-giardin gene was tested on 21 Giardia duodenalis reference strains representing Assemblages A, B and E, which are associated with infections of humans and other mammals. The assay was then applied to 30 faecal samples collected from Italian persons. The sequence analysis of 31 PCR products from both reference strains and clinical samples showed that each Assemblage is clearly distinct from the others on the basis of specific substitutions; the sequence diversity was approximately 5%, and all substitutions occurred at the third codon positions of the gene. The analysis of the intra-Assemblage variability allowed for the identification of three genotypes within Assemblage A, and of four genotypes within Assemblage B. Interestingly, two genotypes were identified only in the clinical samples and not in reference strains. Finally, a simple PCR-restriction fragment length polymorphism method was developed for the rapid discrimination of Assemblages and applied for the direct genetic analysis of cysts present in human faecal samples.