Abstract: To establish a reliable method for measuring the radiochemical purity of technetium99mTc methylenediphosphonate injection, analysis conditions such as stationary phase and developing solvent were studied. The analytical method was methodologically verified in accordance with the Chinese Pharmacopoeia and other relevant provisions. The results showed that the analytical method of single-system thin-layer chromatography, in which polyamide-6 thin-layer plate was used as the stationary phase, V (1 mol/L sodium acetate solution): V (0.1 mol/L hydrochloric acid solution) =1∶1 mixed solution was used as the developing solvent, could improve the tailing problem of the main peak of technetium99mTc methylenediphosphonate injection. The resolution of radioactive main peak and potential impurities including pertechnetate99mTc and colloidal technetium99mTc was greater than 1.0. The method had a good linear relationship in the radioactivity range of 2.7×10-2 μCi to 42.4 μCi, and the correlation coefficient r was 0.997, with good specificity, good durability and intermediate precision. Compared with the analytical method of the pharmacopoeias, the relative error of radiochemical purity met the requirement of less than 2.0%. The established thin-layer chromatography is suitable for determination of the radiochemical purity of technetium99mTc methylenediphosphonate injection.