Two extrachromosomal double-stranded RNA (dsRNA) elements occur in Cryptosporidium parvum. A heteroduplex mobility assay (HMA) was developed for the rapid characterization of sequence diversity in a 173-bp fragment of the small dsRNA element of Cryptosporidium with either a natural sequence from Cryptosporidium meleagridis or a synthetic sequence as reference DNA. The 173-bp fragment was generated from 265 samples of whole feces (242 from humans and 18 from livestock with C. parvum genotype 1 or 2, 4 from humans with Cryptosporidium felis, and 1 from a human with C. meleagridis). The HMA method identified 21 patterns in C. parvum (8 in genotype 1, 12 in genotype 2, and a type common to both genotypes), 4 patterns in C. felis, and 1 pattern in C. meleagridis. All patterns were confirmed as distinct by DNA sequencing. For genotype 1, a single HMA type was found in 89% of samples: 64 of 65 cases from three waterborne outbreaks, all 16 cases from eight intrafamilial outbreaks, and 17 of 28 sporadic cases. Among the remaining 11 sporadic cases due to genotype 1, seven other HMA types were detected. For genotype 2, a single HMA type was found in 72% of samples: 36 of 43 cases from three waterborne outbreaks, 11 of 15 cases from seven intrafamilial outbreaks, 44 of 75 sporadic cases, and all 18 samples from livestock. Within the intrafamilial outbreaks, two other HMA types were identified: the same HMA type was detected in samples from cases within the same outbreak. Among the sporadic cases due to genotype 2, 10 additional HMA types were detected.