The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established gamma-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1-3 h of in vitro radon exposure, a specific pattern of gamma-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of gamma-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and gamma-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, gamma-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of gamma-H2AX foci after in vitro radon exposure and the ratio of RBM-to PBL-absorbed doses in rats following radon exposure.