PURPOSE: Cryptosporidium spp. and Giardia duodenalis are zoonotic protozoan parasites that are widely seen in domestic and wild animals worldwide. While these pathogens, which affect the digestive system of the hosts, cause high economic losses in animal breeding, they are also considered an important public health problem. In recent years, molecular-based studies revealed that 120 genotypes belonging to 44 Cryptosporidium species and eight G. duodenalis assemblages (G. duodenalis A-H) circulate among hosts. The aim of the study was to determine the presence and prevalence of cryptosporidiosis and giardiosis in buffaloes, for which there was only one previous study on the subject in Türkiye. METHODS: In this study, Cryptosporidium spp. and Giardia duodenalis were researched in water buffaloes using polymerase chain reaction (PCR) and DNA sequencing. A total of 510 water buffalo stool samples were obtained from Sivas province, an important water buffalo breeding center in Türkiye. RESULTS: Cryptosporidium spp. were detected in 20 samples (3.92%), whereas five samples (0.98%) were found to be infected with G. duodenalis. DNA sequence analyses of 18S rRNA and β-giardin genes revealed that five Cryptosporidium species, C. occultus (n = 1), C. andersoni (n = 1), C. ryanae (n = 16), C. parvum (n = 1), and C. bovis (n = 1), and G. duodenalis assemblages E were circulated in water buffaloes in Türkiye, respectively. In this work, C. ryanae was the most prevalent Cryptosporidium species, and DNA sequence analyses of these samples showed that 100% nucleotide identities were present between them. Cryptosporidium occultus (PP754270), C. andersoni (PP754271), C. ryanae (PP754272-PP754279, PP754281-PP754285, PP754287-PP754289), C. parvum (PP754280), and C. bovis (PP754286) obtained from water buffaloes in this study shared 98.59-100%, 99.88-100%, 99.49-100%, 99.62-100%, and 99.87-100% nucleotide similarity with isolates present in GeneBank, respectively. In addition, G. duodenalis (PP798352-PP798356) isolates had 99.56-100% (β-giardin) nucleotide identities with G. duodenalis isolates. CONCLUSION: The existence of cryptosporidiosis (the five species) in water buffaloes was reported for the first time in the country. Moreover, one species (C. occultus) has been reported for the first time in Türkiye.