The functional mitochondrial protein alternative oxidase (AOX) gene was used as a marker to analyse the phylogenetic relationship between Cryptosporidium isolates. This gene was characterized, and the phylogentic tree was established from Cryptosporidium isolates and compared to those generated from 18S rRNA and heat-shock protein 70 (HSP70) gene sequences. The present trial aimed at finding out whether the AOX gene is suitable for phylogenetic analysis of the Cryptosporidium genus. The results revealed that the genus Cryptosporidium contained the phylogenetically distinct species C. parvum, C. hominis, C. suis and C. baileyi, which were consistent with the biological characterization and host specificity reported earlier. Cryptosporidium species formed two clades: one included C. hominis, C. suis, C. parvum cattle genotypes and C. parvum mouse genotype; and the other comprised C. meleagridis and C. baileyi isolates. Within C. parvum, both the mouse genotype and the pig genotype I (also known as C. suis) isolates differed from cattle and human (also known as C. hominis) genotypes, based on the aligned nucleotide sequences. The sequence identity of the AOX gene was higher between C. meleagridis and C. baileyi than between C. meleagridis and C. parvum. The phylogenetic trees showed that C. meleagridis was closer to C. baileyi than to C. parvum. This result was inconsistent with the phylogenetic analysis deduced from 18S rRNA and HSP70 gene sequences, respectively. The present results suggest that the AOX gene is not only equally suitable for the phylogenetic analysis of Cryptosporidium, but also provides an outstanding and new approach in determining Cryptosporidium heredity.