Background: Rickettsial diseases are considered important in public health due to their dispersal capacity determined by the particular characteristics of their reservoirs and / or vectors. Among the latter, fleas play an important role, since the vast majority of species parasitize wild and invasive rodents, so their detection is relevant to be able to monitor potential emerging diseases. The aim of this study was to detect, characterize, and compare Rickettsia spp. from the fleas of micromammals in areas with different human population densities in Chile.Methods: The presence of Rickettsia spp. was evaluated by standard polymerase chain reaction (PCR) and sequencing in 1,315 fleas collected from 1,512 micromammals in 29 locations, with different human population densities in Chile. A generalized linear models (GLM) was used to identify the variables that may explain Rickettsia prevalence in fleas. Results: DNA of Rickettsia spp. was identified in 13.2% (174 of 1,315) of fleas tested. Fifteen flea species were found to be Rickettsia-positive. The prevalence of Rickettsia spp. was higher in winter, semi-arid region and natural areas, and the infections in fleas varied between species of fleas. The prevalence of Rickettsia among flea species ranged from 0%–35.1%. Areas of lower human density have the highest prevalence of Rickettsia. The phylogenetic tree shows two well-differentiated clades. Rickettsia bellii is positioned as basal in a clade. Another clade is subdivided into two subclades, and are related to Rickettsia of spotted fever group.Conclusions: To our knowledge, this is the first report of the occurrence and molecular characterization of Rickettsia spp. in 15 species of fleas of micromammals in Chile. In this study, fleas were detected carrying Rickettsia DNA with zoonotic potential, mainly in villages and natural areas of Chile. Considering that there are differences in the prevalence of Rickettsia in fleas associated with different factors, more investigations are needed to further understand the ecology of Rickettsia in fleas and their implications for human health.