Prostate-specific membrane antigen (PSMA) is a well validated prostate cancer marker, but reported PSMA-targeted tracers derived from the Lys-urea-Glu pharmacophore including the clinically validated [99mTc]Tc-EDDA/HYNIC-iPSMA have high off-target uptake in kidneys, spleen and salivary glands. In this study, we synthesized and evaluated three novel 99mTc-labeled PSMA-targeted tracers and investigated if the tracers derived from the Lys-urea-Aad pharmacophore could have minimized uptake in off-target organs/tissues. In vitro competition binding assays showed that compared with HYNIC-iPSMA, the three novel ligands had slightly weaker PSMA binding affinity (average Ki = 3.11 vs 8.96 - 11.6 nM). Imaging and ex vivo biodistribution studies in LNCaP tumor-bearing mice showed that [99mTc]Tc-EDDA/HYNIC-iPSMA and the three novel tracers successfully visualized LNCaP tumor xenografts in SPECT images and were excreted mainly via the renal pathway. The average tumor uptake at 1 h post-injection varied from 5.40 to 18.8 %ID/g, and the tracers derived from the Lys-urea-Aad pharmacophore had much lower uptake in spleen and salivary glands. Compared with the clinical tracer [99mTc]Tc-EDDA/HYNIC-iPSMA, the Lys-urea-Aad derived [99mTc]Tc-EDDA-KL01127 had lower background uptake and superior tumor-to-background contrast ratios, and is therefore promising for clinical translation to detect prostate cancer lesions with SPECT.