Hammondia heydorni is regarded as a protozoan parasite that uses canids, e.g. dogs and foxes, as definitive hosts, but clinical signs of infection are rare. This study therefore took advantage of the opportunity to study an oocyst population from the faeces of a dog suffering from intermittent bouts of diarrhoea. Oocysts from the naturally infected dog were shown to be H. heydorni by using the polymerase chain reaction combined with DNA sequencing as a diagnostic tool.The nucleotide sequence data reported in this paper are available from GenBank under the following Accession numbers DQ183058, DQ183059 and DQ022687. A comparison of the first internal transcribed spacer (ITS1) sequence of ribosomal DNA obtained with those from other dog and fox oocysts, previously regarded as H. heydorni, showed these oocysts contained identical ITS1 sequences. However, the oocyst DNA from the fox and dog differed by the presence/absence of a 9 bp insertion/deletion within intron 1 of the alpha tubulin gene, and this difference was conserved across a number of different oocyst populations from the 2 species of host. A PCR assay was established that takes advantage of this insertion/deletion and is able to differentiate between the 2 oocyst populations. This study therefore provides evidence that H. heydorni oocysts from dogs and foxes represent 2 distinct genetic lineages that can be differentiated using a PCR, which targets the alpha tubulin locus.