© 2023 by the authors.Giardia duodenalis (G. duodenalis) is an important zoonotic protozoan agent that causes foodborne and waterborne diarrhea in humans and other mammals. Molecular-based tests are critical in diagnosing giardiasis in humans and animals, identifying species, understanding the zoonotic potential and transmission routes, and evaluating taxonomy. Therefore, this study aimed to investigate the molecular characterization of G. duodenalis in buffaloes in the Van region in Türkiye. Buffaloes are a species that has been poorly studied in this regard. For this purpose, 100 fecal samples were collected from buffaloes in the Van region. The DNA extraction was performed using the GeneMATRIX STOOL DNA Purification Kit from stool samples. The nested PCR test was performed with the appropriate primers from the obtained DNA samples. The obtained bands suitable for sequencing were sent for sequence analysis, and the sequence results were aligned bidirectionally and compared with the database of GenBank by BLAST. As a result of the study, an 11% positivity rate for G. duodenalis was found in buffaloes, and assemblage E and assemblage B were isolated. To our knowledge, assemblage B in buffaloes was reported for the first time in this study. As a result, it was concluded that buffaloes are an important reservoir for waterborne and foodborne giardiasis.