A seroprevalence study for IgG antibodies against spotted fever group (SFGR) and typhus group (TGR) Rickettsia among humans and domestic pets was conducted in the city of Iquitos, located in the Amazon basin of Peru. Of 1,195 human sera analyzed, 521 (43.6%) and 123 (10.3%) were positive for SFGR and TGR antibodies, respectively. District of residence and participant age were associated with antibody positivity for both groups, whereas rodent sightings in the home were associated with TGR antibody positivity. Of the 71 canines tested, 42 (59.2%) were positive for SFGR antibodies, and two (2.8%) were positive for TGR antibodies; one active SFGR infection was detected by polymerase chain reaction. An uncharacterized SFGR species was detected in 95.9% (71/74) of Ctenocephalides felis pools collected from domestic pets. These data suggest that rickettsial transmission is widespread in Iquitos. Rickettsia species should be further explored as potential causes of acute febrile illnesses in the region. Copyright © 2010 by The American Society of Tropical Medicine and Hygiene.