Our aim was to determine the presence of Rickettsia spp. in 66 fleas from Uruguay. Rickettsial DNA was amplified using gltA and ompB PCR primers. Rickettsia spp. were found in 41% of the fleas (25 Ctenocephalides felis and 2 Ctenocephlides canis). Sequences resulted in the identification of Rickettsia felis and four genotypes closely related to this species (Rickettsia sp. TwKM03, California 2, Hf187, and RF2125). The presence of R. felis in fleas from Uruguay in was demonstrated. This is the second species of Rickettsia identified in Uruguay in the past 2 years using molecular approaches, and it is helping to clarify the etiology of rickettsial diseases in the region.