Abstract: To develop novel technetium-99m-labeled thymidine analog for tumor SPECT imaging, two ⁹⁹Tc^m-labeled thymidine analogs (⁹⁹Tc^m-ANMdU and ⁹⁹Tc^m-NHT) were synthesized. ⁹⁹Tc^m-ANMdU and ⁹⁹Tc^m-NHT are stable in aqueous solution and radiochemical purity are over 95%. Biodistribution of ⁹⁹Tc^m-ANMdU in tumor-bearing mice shows that the high uptake of ⁹⁹Tc^m-ANMdU in kidney and the clearance is fast. At 60min p.i., the ratios of tumor-to-muscle, tumor-to-bone and tumor-to-blood are 1.58±0.17, 1.95±0.31 and 1.17±0.08, respectively. Biodistribution of ⁹⁹Tc^m-NHT in mice shows that the high uptake of ⁹⁹Tc^m-NHT in liver and kidney, indicating that the clearance of ⁹⁹Tc^m-NHT is mainly through the hepatobiliary pathway and the renal pathway. At 2 h p.i., the ratios of tumortomuscle, tumor-to-bone and tumor-to-blood are 4.41±0.32, 2.45±0.24 and 1.51±0.18, respectively. These indicate that ⁹⁹Tc^m-NHT is probably a potential SPECT tumor imaging agent and further study is needed.