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Purpose To evaluate radiation induced chromosome aberration frequency in peripheral blood lymphocytes of cosmonauts who participated in flights on Mir Orbital Station and ISS (International Space Station). Materials and methods Cytogenetic examination which has been performed in the period 1992–2008 included the analysis of chromosome aberrations using conventional Giemsa staining method in 202 blood samples from 48 cosmonauts who participated in flights on Mir Orbital Station and ISS. Results Space flights led to an increase of chromosome aberration frequency. Frequency of dicentrics plus centric rings (Dic+Rc) depend on the space flight duration and accumulated dose value. After the change of space stations (from Mir Orbital Station to ISS) the radiation load of cosmonauts based on data of cytogenetic examination decreased. Extravehicular activity also adds to chromosome aberration frequency in cosmonauts’ blood lymphocytes. Average doses after the first flight, estimated by the frequency of Dic+Rc, were 227 and 113 mGy Eq for long-term flights (LTF) and 107 and 53 mGy Eq for short-term flights (STF). Conclusion Cytogenetic examination of cosmonauts can be applied to assess equivalent doses.