ABSTRACT. Cattle are major hosts of Cryptosporidium. Cryptosporidiosis in neonatal calves is associated with retarded growth, weight loss and calf mortality, and zoonotic infections in humans. Fecal samples were collected from calves in Ishikari District, Hokkaido, Japan and examined by PCR and sequence analyses. Among the 107 fecal samples collected in May and June 2012, 25 (23%) were positive for Cryptosporidium, including 8 samples (7%) having C. parvum, 10 (9%) having C. bovis and 7 (7%) having C. ryanae. This is first time C. ryanae has been detected in Hokkaido. Furthermore, it is the first detection of C. ryanae from pre-weaned calves in Japan. Microscopic observation with the flotation method is powerful and traditional tool for screening for Cryptosporidium species, but it sometimes leads to low detection of Cryptosporidium with low oocyst shedding intensity. If calves with or without diarrhea are examined using the molecular diagnostic tools, C. bovis and C. ryanae might be detected in other areas of Japan including Hokkaido. Here, the zoonotic species, C. parvum, was also observed. Therefore, calves can be potential sources of cryptosporidial infections for humans and other animals. The detection of C. parvum was statistically correlated with diarrhea in calves.