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Folate conjugates exhibit high affinity for folate receptor (FR) positive cells and tissues, such as those in tumors, making them attractive candidates and of interest in diagnostic tumor imaging. The aim of study was to synthesize a novel radiopharmaceutical based folate conjugate, 99mTc-citro-folate, and to evaluate its efficiency as a targeted agent for imaging tumors that over express FR. TLC, HPLC 1H NMR and LC–MS/MS methods were used to check and confirm the synthesized citro-folate. Citro-folate was labeled with Tc-99m with high labeling efficiency (97 ± 1.0%). Biodistribution of the radiolabeled conjugate 99mTc-citro-folate was investigated in vivo using two groups of rats: FR saturated and unsaturated. These experiments showed high uptake of 99mTc-citro-folate in FR rich tissues and demonstrated its sensitivity and specificity in imaging ovary and uterus. Based on the demonstrated good radiolabeling and biodistribution properties, the compound 99mTc-citro-folate may potentially be used as a radiopharmaceutical agent for imaging the FR-positive tumors.