Cryptosporidium is a protozoan parasite causing gastrointestinal illness. Drinking waterborne outbreaks have been caused by C. hominis, C. parvum and C. cuniculus. Molecular detection techniques already exist for Cryptosporidium and usually target housekeeping genes. We set ourselves the task to identify species-specific genes. These genes are likely to be involved in host parasite interaction and virulence. Three subtelomeric species-specific putative virulence factor genes (Cops-2, Chos-1 and Chos-2) were identified in silico and used to develop novel real-time PCR assays. Our results show that Chos-2 is a suitable target for probe-based assays for the specific detection of C. hominis and C. cuniculus (two very closely related species) and that Cops-2 is a suitable target for specific detection of C. parvum.