Dermacentor marginatus species is the common vector of Rickettsia slovaca. Recently, a new rickettsia genotype, RpA4, was detected in several Dermacentor sp. ticks in several countries of the former Soviet Union and in Spain. In an epidemiological surveillance study, ninety-eight D. marginatus, collected in the South of Portugal in spring of 2003 and 2004, were screened by polymerase chain reaction (PCR) to detect Rickettsiae. Overall, 59.2% of the ticks were PCR-positive, among them 34.5% were proved to be infected with R. slovaca and 65.5% with Rickettsia sp. RpA4. This study reports for the first time a high prevalence of Rickettsia sp. RpA4 in D. marginatus in Portugal, pointing out for a wider geographic distribution of this genotype than previously reported.