Rickettsioses, often underreported, pose public health challenges. Rickettsia asembonensis is a potential emerging pathogen that was previously detected in humans, animals, and a variety of arthropods. While its pathogenicity in humans remains unclear, it poses a potential public health threat. Here, we present an extended epidemiological, diagnostic, and genetic analysis of the information provided in a preliminary report on the investigation of rickettsiae in Peru. In particular, we report the detection of R. asembonensis in blood specimens collected from four human patients with an acute undifferentiated fever of a seven- to nine-day duration, all of whom tested negative for other vector-borne pathogens. Additionally, we describe the replicative capacity of the R. asembonensis isolates in cell cultures.