A total of 1,138 blood specimens were collected over a 6-year period (2000-2006) from domestic dogs in South Africa. Specimens from domestic dogs were obtained from the Onderstepoort Veterinary Academic Hospital (OVAH) in Pretoria, the Society for the Prevention of Cruelty to Animals (Johannesburg, Durban, East London and Bloemfontein) and private practices from four provinces (Gauteng, Mpumalanga, KwaZulu-Natal and Western Cape). All specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. On RLB, 560/1,137 domestic dog-specimens were positive for one or more parasites. Of the positive domestic dog-specimens, 420 (75%) were infected with Babesia rossi; 82 (15%) dogs were infected with Theileria sp. (dog); 18 (3%) dogs were infected with Babesia vogeli; 14 (3%) specimens were infected with Ehrlichia canis. Mixed infections were also found: B. rossi and E. canis were detected in 12 (2%) specimens; B. vogeli and E. canis occurred in 7 (1%) specimens; Theileria sp. (dog) and E. canis in 3 (0.5%) specimens; B. rossi and B. vogeli in one specimen. B. rossi, B. vogeli and E. canis occurred simultaneously in one dog. There was also one incidental finding of a dog positive for Trypanosoma congolense. The results indicate that a wide range of tick-borne pathogens are circulating in the canine populations in South Africa.