Cryptosporidium oocysts were detected in the feces of cattle in Saga, Japan. Isolates were morphologically large. We attempted to identify the species or genotypes of the isolates by analyzing the partial sequences of the 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes, and measuring the infectivity in mice. The isolates showed 100% homology with Cryptosporidium andersoni in the COWP gene sequence and it could be transmitted to mice, but in the 18S rRNA gene, there was an additional signal in the ABI sequence chromatogram. To examine the additional signal, we analyzed both the 18S rRNA and the COWP gene sequences of a single oocyst passaged from mice using a modified multiplex PCR that was able to amplify both genes. As a result, it was revealed that two distinct genotypes (Types A and B) of a novel C. andersoni type existed in the 18S rRNA gene, whereas the COWP gene sequences of both oocysts were identical to C. andersoni. Although the sequence of the 18S rRNA gene of Type A was identical to that of C. andersoni, that of Type B had a thymine insertion and was not identical to any sequence registered with GenBank. Here we report that this is a new type of C. andersoni.