In northwestern Mexico, studies on hantavirus in rodent hosts are scarce. Our objective was to conduct serological tests to detect antibodies against hantavirus in rodents from the Janos–Nuevo Casas Grandes prairie dog complex (JCGC) in northwestern Mexico. In December 2007 and April 2008, we captured 149 rodents and tested for immunoglobulin G antibodies to New World hantaviruses. Three Peromyscus maniculatus from grassland habitats without prairie dog colonies were antibody positive. This is the first record of hantavirus prevalence in wild rodents from JCGC and from the state of Chihuahua. Further molecular analysis is needed to describe which hantavirus is circulating in this area.