The advent of the polymerase chain reaction (PCR) coupled with the specificity of deoxyribocucleic acid (DNA)-DNA hybridization has led to the development of specific and sensitive molecular diagnostic tests to detect and characterize the organisms that cause theileriosis and heartwater. Theileriosis is a widespread disease of wild and domestic ruminants caused by apicomplexan parasites of the genus Theileria. Species-specific variations in small subunit ribosomal ribonucleic acid genes (SSUrRNA) have been used to develop probes that can distinguish between Theileria species such as T. parva, T. annulata, T. mutans, T. buffeli and T. taurotragi. Routine application of this test has led to the discovery of previously unknown species, such as Theileria sp. (buffalo) which is apparently apathogenic to both buffalo and cattle, and Theileria sp. (sable) which is pathogenic to sable and possibly also to roan antelope. In addition, characterization probes located in the internal transcribed spacer (ITS) can be used to distinguish between most isolates of the causative agents of East Coast fever (T. p. parva) and Corridor disease (T. p. lawrencei). Heartwater is an economically important disease of livestock and some wild ruminants, caused by the intracellular rickettsial parasite Ehrlichia (ex Cowdria) ruminantium. DNA probes used to detect and characterize E. ruminantium isolates include SSUrRNA (16S) probes, the pCS20 probe and map1 probes. A panel of eight 16S probes has been developed for the detection of E. ruminantium and related Ehrlichia species. There are probes for 5 different E. ruminantium genotypes, one which will detect all 5 of these genotypes, one to detect any Ehrlichia species other than E. ruminantium, and one for any Anaplasma species. The pCS20 probe is specific for E. ruminantium and is the most sensitive of the probes for E. ruminantium detection, but it is not able to distinguish among the different genotypes. The map1 gene has also been used for diagnosis, but the extensive polymorphism of this gene means that it is most useful for characterization of different genotypes of the parasite. Routine application of these tests has led to the discovery of new genotypes that are probably not E. ruminantium but are probably new species of Ehrlichia.