Restriction fragment length polymorphism (RFLP) analysis of isolates of Cryptosporidium parvum has revealed two subgroups, termed H and C. The limited resolution of the RFLP method precludes an in-depth study of the genetic structure of C. parvum populations. Published C. parvum restriction polymorphisms lie within protein-coding regions known to be more homogeneous than noncoding sequences. To better assess the degrees of heterogeneity between and within C. parvum isolates, sequence polymorphism in the beta-tubulin intron, the only C. parvum intron described to date, was investigated. In contrast to the two genotypes distinguished by multilocus RFLP, several alleles were detected by sequence and RFLP analysis of the beta-tubulin intron and adjacent exon 2. Isolates carrying different beta-tubulin alleles were found. Significantly, one of the beta-tubulin alleles present in two geographically unrelated isolates combined features of C- and H-type isolates, suggesting that it might have arisen from a recombination event. A comparison of multiple samples of a calf-propagated laboratory isolate showed that the ratio of different beta-tubulin alleles fluctuated during serial passage.