Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m (99mTc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). 99mTc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for 99mTc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with 99mTc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n = 5) received no blockade; Group II (n = 5) received a blocking dose of non-radiolabeled TCP-1. Group III (n = 5) were imaged with 99mTc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n = 5) were also imaged with 99mTc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of 18F-fluorodeoxyglucose (18F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro99mTc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04 ± 0.52 nM. In cancer xenografts, 99mTc-TCP-1 radioactivity (%ID/g) was 1.01 ± 0.15 in the absence of blockade and was reduced to 0.26 ± 0.04 (P < 0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with 99mTc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by 18F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions.