Three new genes of Cryptosporidium parvum were cloned, including a gene encoding methionine aminopeptidase, one encoding chaperonin containing T-complex protein 1 delta (TCP-1 delta) and one with unknown function. DNA sequence analysis indicated that these genes are quite conserved, but there were some base pair differences between genotype I and genotype II isolates. These differences were confirmed by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 3 genes from 41 isolates collected from different hosts and geographical origins. In brief, the band patterns generated by endonuclease Hind III or Hinf I restrictions of the gene of methionine aminopeptidase, Sac I restriction of the gene of chaperonin, or Ava II restriction of the unknown gene could differentiate the isolates of C. parvum into genotype I and genotype II. PCR primers based on these genes amplified only C. parvum genes. Even a single oocyst was detectable with these PCR primers. Thus the results provided further evidence that genotype I and genotype II are distinct, and our three new primers can be used to detect and characterize C. parvum isolates with high sensitivity.