Sequence analysis of a fragment of the beta-tubulin gene was performed on 13 isolates of the parasite Cryptosporidium parvum, eight from humans and five from animals. A total of 12 synonymous substitutions and a deletion of two bases within the intron sequence were found. This genetic variation defined two alleles at the beta-tubulin locus, which can be identified by a simple polymerase chain reaction-restriction fragment length polymorphism assay. A total of 20 isolates were also tested using four available molecular markers. These analyses showed congruently that the C. parvum isolates segregate into two groups, one found exclusively in humans and the other found in both humans and animals. Since no recombinant genotypes were observed, the results are consistent with the hypothesis of a substantially clonal reproduction in this parasite.