α v β 6 integrin is overexpressed by several carcinomas and thus considered a target for diagnostic imaging and anticancer therapies. Recently, we presented the α v β 6 integrin-binding peptide SFITGv6 as a novel potential tracer for imaging and targeted therapy of α v β 6 integrin–positive carcinomas. Here, we analyzed the affinity and specificity of 5 native α v β 6 integrin–specific binders in comparison to SFITGv6. Methods: Sunflower trypsin inhibitor 1 (SFTI1)–based peptides containing arginine-glycine-aspartic acid (RGD) motif-spanning octamers of fibronectin (SFFN1), tenascin C (SFTNC), vitronectin (SFVTN), and latency-associated peptides (LAP) 1 (SFLAP1) and 3 (SFLAP3) were synthesized, and their binding potential to α v β 6 integrin–expressing head and neck squamous cell carcinoma (HNSCC) cell lines was evaluated. Subsequently, stability, affinity, and specificity were assessed in vitro using radio–high-pressure liquid chromatography, surface plasmon resonance assay, and binding experiments including competition, kinetics, internalization, and efflux. α v β 6 integrin binding specificity was further evaluated by peptide histochemistry. Finally, in vivo binding properties were assessed using small-animal PET imaging and biodistribution experiments in HNSCC-bearing mice, and 68 Ga-DOTA-SFLAP3 was applied for diagnostic PET/CT of an HNSCC patient. Results: When the newly designed peptides were compared, significant binding (〉20%) to several HNSCC cell lines (HNO97, HNO399, and HNO223) and a fast internalization of up to 60% and 70% were observed for SFLAP3 (GRGDLGRL) and SFITGv6 (FRGDLMQL). In contrast, the other peptides displayed binding that was moderate (SFLAP1, 4.1%–12.1%) to marginal (SFFN1, SFTNC, and SFVTN, 〈1%) and were therefore excluded from further analysis. Notably, SFLAP3 exhibited improved affinity for α v β 6 integrin (mean half-maximal inhibitory concentration, 3.5 nM; dissociation constant, 7.4). Moreover, small-animal PET imaging and biodistribution studies of HNSCC xenograft mice revealed an increased tumor-specific accumulation 30–60 min after injection of 68 Ga-labeled or 177 Lu-labeled DOTA-SFLAP3. Peptide staining further demonstrated binding specificity for SFLAP3 to HNSCC tumor cells. Finally, PET/CT scanning of an HNSCC patient showed specific SFLAP3 accumulation in the primary tumor lesion (SUV max , 5.1) and in corresponding lymph node metastases (SUV max , 4.1). Conclusion: SFLAP3 represents a promising tracer for prognostic assessment, diagnostic imaging, and possibly targeted therapy of α v β 6 integrin–expressing tumors.