A 4-nitroimidazole isocyanide derivative (6) was synthesized and radiolabelled with 99mTc(CO)3 core in high yield. It was interesting to note that 99mTc(CO)3-6a and 99mTc(CO)3-6b can be prepared at 100 °C and 25 °C, respectively. 99mTc(CO)3-6a had three 6 molecules while 99mTc(CO)3-6b contained two 6 molecules. The corresponding rhenium complexes were prepared to confirm the structure of the 99mTc complexes. Both complexes showed good stability in vitro and hypoxic selectivity. The partition coefficient results indicated both of them were hydrophilic and 99mTc(CO)3-6a was more hydrophilic than 99mTc(CO)3-6b. From the biodistribution study results, 99mTc(CO)3-6a showed higher tumor/blood and tumor/muscle ratios at 2 h post-injection. Further, single photon emission computed tomography (SPECT) imaging study of 99mTc(CO)3-6a showed there was an observable tumor uptake, suggesting it would be a potential tracer for imaging of tumor hypoxia.