Cryptosporidium parvum is an enteric protozoan parasite causing intestinal illness in humans world-wide. Identification of polymorphic genetic loci would aid investigators in tracing the origin of contaminating oocysts, and accurately assess the main routes of transmission. A new DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was used to detect genetic variability among ten C. parvum isolates. Eight isolates of animal origin generated identical fingerprint patterns with each of nine primer pairs tested. The same patterns were produced by a human isolate passaged in calves. However, unique patterns were generated by an isolate obtained directly from human feces. A PCR assay, targeting a TGA polymorphism within the 18S rRNA gene, was also used to identify genetic heterogeneity among the ten isolates. Segregation of the isolates using this PCR assay highly correlated with AFLP analysis. Additional studies with a sequence-specific PCR assay, targeting regions of the 18S rRNA gene, revealed that the ten isolates shared a common 18S ribosomal RNA genotype.