Rickettsia massiliae, a member of the spotted fever group of Rickettsia, was first isolated from a Rhipicephalus turanicus tick in France. In the New World, it has been detected in Rhipicephalus sanguineus ticks from different geographical locations in Argentina and the United States, but it has only been isolated in Arizona. The aim of this study was the isolation and genetic characterization of R. massiliae from R. sanguineus ticks collected from dogs in Buenos Aires city, Argentina. In total, 49 R. sanguineus ticks were collected from 10 dogs and grouped into 10 pools of 4–5 specimens. With a PCR assay, which detects a fragment of the Rickettsia genus-specific 23S-5S intergenic space, 1 pool of 5 ticks was found positive. Generated sequences exhibited 100% identity with R. massiliae. A new isolate, named CABA, was obtained from this pool by inoculating it into monolayers of Vero cells. Genotypic characteristics were determined, and results showed that fragments of the 23S-5S intergenic space, ompA, ompB, gltA, htrA, and sca1 genes had great similarity with R. massiliae strain Bar29 (Spain). Although few human cases have been confirmed for this pathogen, its circulation in urban areas is of great importance to public health. This isolation improves knowledge of the circulating pathogen and could improve future diagnostic processes as it allows the production of more specific antigens for serological testing.