The work was carried out in the reserve zone on the territory of the Il’men State Reserve in the belt of pine-birch forests in the Southern Ural (Chelyabinsk region) during 2013–2014. A targeted study of rodents with splenomegaly for their infection with pathogens of natural focal infections as a possible cause of the phenomenon studied was carried out. Modern virological and bacteriological methods of laboratory analysis, including using PCR-test systems were used. Hemorrhagic fever, borreliosis and monocytic erlichiosis was found at representatives of different families of the order Rodentia (Cricetidae and Muridae). The obtained data testify to the reservoir role of the rodent population in the carriage of borreliosis and hemorrhagic fever (46 % and 26 % of animals, respectively). According to the data of the Federal Surveillance Agency of the Federal Service for Consumer Protection and Consumer Rights of the Federal Service for Supervision of Consumer Rights and Social Protection of the Russian Federation for the Ural Federal District, a similar level (32 %) of the infection of rodents with hantavirus infection was registered in the forest-steppe region of the Chelyabinsk region during the same period. Differences in the proportion of infected individuals of background species were noted. The relationship between splenomegaly and rodent infection is discussed.