Molecular diagnostic assays for Cryptosporidium are usually based on polymerase chain reaction (PCR) and may detect the entire genus or target-specified species. Of the ever-growing number of species, fewer than half have been reported from humans, and most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or Cryptosporidium hominis. Here, we describe a real-time PCR for the detection of all Cryptosporidium spp. that can then be differentiated by sequencing the PCR amplicons and a duplex real-time PCR for the simultaneous detection and differentiation of C. parvum and C. hominis.