Ticks can transmit multiple pathogenic bacteria responsible for diseases in animals and humans such as Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and spotted fever group Rickettsia sp. The current study aimed to investigate the presence of Rickettsiae in ticks collected from seven localities in Serbia. One hundred thirty-one (131) questing ticks belonging to 5 tick species (Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Haemaphysalis Concinna, and Ixodes ricinus) were collected in 2007 and 2009. Ticks were tested by polymerase chain reaction, amplifying gltA, ompA, and 17-kd genes, and sequencing analysis, revealing the presence of Rickettsia helvetica and Rickettsia monacensis in I. ricinus ticks only (infection rates 7.7% and 15.4% for R. helvetica and R. monacensis, respectively). R. helvetica has been isolated from I. ricinus ticks and has been implicated in fatal perimyocarditis. R. monacensis was first identified in I. ricinus samples collected in Germany and has recently been implicated in human infection. The results of the current study make fundamental the need to evaluate the incidence of infection with R. helvetica and R. monacensis among the resident population.