Technical Abstract: Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. New diagnostic genetic loci, based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), were developed to aid in the detection of H. heydorni-like parasites and to discriminate them from N. caninum and other related coccidians of dogs. Based on the data obtained from 5 random decamers, H. heydorni (Manhattan-1) and N. caninum (NC1) were characterized by distinct banding patterns (similarity index = 0.068). High stringency PCR assays were developed from the sequences of 2 cloned bands (Genbank accession numbers BZ592549 and BZ592593), uniquely amplified from H. heydorni. Interestingly, using these primers, PCR amplification was achieved only from 2 of the 5 isolates presumed to represent H. heydorni. The same result was obtained from these 5 isolates using a recently described PCR assay directed to the H. heydorni internal transcribed spacer (ITS)-1. It is concluded that H. heydorni and N. caninum are genetically distinct and that such tools may be useful for more detailed characterization of the diversity of related parasites occurring in dogs.