Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.