Cryptosporidiosis is an important zoonosis caused by the Cryptosporidium species. To develop a PCR diagnostic kit for molecular detection and differential diagnosis of Cryptosporidiurn spp., a portion of ITS-1 sequence of Cryptosporidium. Andersoni was chosen as the target DNA for designing the species-specific primers (ZRQF/ZR). The kit components were determined after the PCR amplification conditions were serially optimized. A series of tests were conducted in the specificity, sensitivity, stability, reproducibility, and stored period of the kit, respectively. The results showed that only C. Andersoni were amplified specific band of about 500 bp, while Cryptosporidium. Parvum, Cryptosporidium. Baileyi, Eimeria sp of dairy cow, Toxoplasma gondii, Eimeria sp of pig, Ascaris suum, Cyclospora sp, and E. Coli could not be amplified. 254 oocysts of C. Andersoni was the lowest number that could be detected using the kit. The kit worked well after being stored at room temperature, 4 and -20℃ for nine months. Fecal specimens, which were collected from a total of 243 calves on four different dairy farms in Guangdong Province, China, and one dairy farm in Henan Province, China, were examined using the kit; the positive rate of the kit was 2-13% higher than that of the routine methods. The results indicated that the kit can detect fecal samples faster, more sensitively, and conveniently, and can provide a useful tool for the identification and differentiation of C. Andersoni from the other Cryptosporidium species; it also has implications for further studies on molecular epidemiology and differential diagnostics of cryptosporidiosis in animals.