The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules ⁶⁴Cu-DOTA-Z_EGFR:1907 and ¹⁸F-FBEM-Z_EGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with ⁶⁴Cu, ¹⁸F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of ¹⁸F-FBEM-Z_EGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of ¹⁸F labeled affibody probes (Al¹⁸F-NOTA-Z_EGFR:1907 and ¹⁸F-CBT-Z_EGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating Z_EGFR:1907 with NOTA and radiolabeling with Al¹⁸F to produce Al¹⁸F-NOTA-Z_EGFR:1907. In a second approach the prosthetic group ¹⁸F-labeled-2-cyanobenzothiazole (¹⁸F-CBT) was conjugated to Cys-Z_EGFR:1907 to produce ¹⁸F-CBT-Z_EGFR:1907. Binding affinity and specificity of Al¹⁸F-NOTA-Z_EGFR:1907 and ¹⁸F-CBT-Z_EGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al¹⁸F-NOTA-Z_EGFR:1907 or ¹⁸F-CBT-Z_EGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al¹⁸F-NOTA-Z_EGFR:1907 and ¹⁸F-CBT-Z_EGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although ¹⁸F-CBT-Z_EGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al¹⁸F-NOTA-Z_EGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al¹⁸F-NOTA-Z_EGFR:1907 and ¹⁸F-CBT-Z_EGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for ¹⁸F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al¹⁸F-NOTA-Z_EGFR:1907 and ¹⁸F-CBT-Z_EGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al¹⁸F-NOTA-Z_EGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.