Shikonin was isolated from Ratanjot pigment then the obtained shikonin was well characterized. This study is aimed to optimize radiolabeling yield of shikonin with 99mTc with respect to factors that affect the reaction conditions such as shikonin amount, SnCl2·2H2O amount, reaction time and pH of the reaction mixture. In vitro stability of the radiolabeled complex was checked and it was found to be stable for up to 6 h. Biodistribution studies showed that, 99mTc–shikonin accumulate in tumor sites with higher T/NT than other currently available 99mTc(CO)3-VIP, 99mTc–nitroimidazole analogues and 99mTc–polyamine analogues indicating that shikonin deliver 99mTc to the tumor sites with a percentage sufficient for imaging and can overcome many drawbacks of other radiopharmaceuticals used for tumor imaging.