Abstract: Radioisotope ^99mTc (T_1/2＝6.01 h) is the decay daughter of ⁹⁹Mo, which is a widely used radioisotope in nuclear medicine. It accounts for about 80% of all radioactive isotopes used. Recently, the direct production of ^99mTc on cyclotron accelerators via ¹⁰⁰Mo(p,2n)^99mTc reaction has been the most promising approach with many advantages, such as nonreactors, no highly enriched uranium, less radioactive waste and no nuclear proliferation. This paper reviews the reported several separation strategies for ^99mTc based on cyclotron production, which includes column chromatography, solvent extraction, chemical precipitation and thermochromatography. This work could provide some theoretical reference for the investigation of direct production of 99mTc on cyclotron accelerator in China.