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Objective: Orthohantaviruses are geographically widely distributed and present various clinical manifestations from mild symptoms to the severe form of haemorrhagic fever with renal syndrome (HFRS) in Eurasia. Official registration of HFRS in Kazakhstan started in the year 2000. However, the true prevalence of human infections by orthohantaviruses within Kazakhstan is unknown. The aim of this study was to investigate of the seroprevalence of orthohantavirus infections in patients with fever of unknown origin (FUO) in two regions, Almaty and Kyzylorda region. Methods: Paired serum samples from 802 patients with FUO were screened for the presence of orthohantavirus IgG and IgM antibodies by ELISA. Positive samples were further tested by immunoblotting and indirect immunofluorescence tests (IIFT) to determine the respective orthohantavirus serotypes. Suspected acute serum samples were additionally checked by a RT‐PCR to identify viral RNA. Results: In total 178/802 (22.2%) serum samples reacted with orthohantavirus IgG antibodies and 4/802 (0.5%) with IgM antibodies. All positive samples were tested by immunoblotting which resulted in 2.9% positive samples with IgG antibodies against Puumala (PUUV), Hantaan (HTNV) and Dobrava (DOBV) virus serotypes in Almaty region and 5.4% to PUUV and DOBV serotypes in Kyzylorda region, respectively. In the IFFT, 1.9% positive samples from Almaty and 3.1% from Kyzylorda were confirmed for PUUV and DOBV serotypes. Out of four IgM ELISA positive samples only three were positive against PUUV in the immunoblot and showed weak positive reactivity for the Saaremaa (SAAV), PUUV and HTNV serotypes in the IFFT. Conclusions: This study demonstrates the presence of orthohantavirus infections among patients with FUO in Kazakh regions that were so far considered as non‐endemic. The healthcare system needs to be prepared accordingly in order to be capable of detecting cases and providing adequate management of patients. [ABSTRACT FROM AUTHOR]