Helicobacter felis, Helicobacter bizzozeronii, and Helicobacter salomonis are frequently found in the gastric mucous membrane of dogs and cats. These large spiral organisms are phylogenetically highly related to each other. Their fastidious nature makes it difficult to cultivate them in vitro, hampering traditional identification methods. We describe here a multiplex PCR test based on the tRNA intergenic spacers and on the urease gene, combined with capillary electrophoresis, that allows discrimination of these three species. In combination with previously described 16S ribosomal DNA-based primers specific for the nonculturable "Candidatus Helicobacter suis," our procedure was shown to be very useful in determining the species identity of "Helicobacter heilmannii"-like organisms observed in human stomachs and will facilitate research concerning their possible zoonotic importance.