With the objective of molecularly characterizing rickettsial isolates from humans with non-specific acute febrile syndrome, a cross-sectional descriptive study was conducted, with isolates propagated in Vero ATCC cellular cultures and alternative lines, verifying the viability by means of Indirect Immunofluorescence. Prior to DNA extraction, the gltA gene was amplified by means of conventional PCR, and its sequence was analyzed. Twelve isolates were amplified, five with sufficient DNA so as to sequence them, exhibiting compatibility with R. asembonensis in four, and a close identity with Coxiella burnetti in one. At least three of seven alternative cellular lines showed significant yield in sub-cultures. R. asembonensis was identified in four isolates of humans with non-specific acute febrile syndrome, coming from the regions of Ayacucho, Cajamarca, and Madre de Dios in Peru, and Coxiella burnetti in one coming from the Loreto region.