Background: Fibroblast activation protein (FAP)-expressing cancer-associated fibroblasts (CAF), a major component of tumor stroma, confer treatment resistance, promote local progression, metastasis and immunosuppression. Because FAP is selectively expressed in the tumor stroma of many cancers, radiolabeled small molecule ligands targeting FAP are being explored for their use as pan-cancer theranostic agents. The objective was to establish the spectrum of FAP expression across various cancers by immunohistochemistry (IHC) and to explore whether 68Ga-FAPi-46 PET image biodistribution faithfully reflects tumor FAP expression from resected tumor and non-tumor specimens. Methods: This study was a prospective, exploratory, imaging trial in cancer patients. Referred volunteer patients scheduled to undergo surgical resection of the primary tumor and/or metastases were eligible. Patients underwent one whole body 68Ga-FAPi-46 PET/CT scan. Subsequently, patients underwent surgical resection of the primary tumor and/or metastasis. The outcome measure was the correlation of 68Ga-FAPi-46 PET maximum standardized uptake value (SUVmax) with FAP IHC score in patient-matched cancer and non-cancer tissue. Results: The frequency of FAP expression across 14 cancers on tissue microarrays ranged from 25 to 100% (mean 76.6±25.3%). For imaging and IHC correlation, fifteen patients with the following cancer types were prospectively included: colorectal (n = 4), head and neck (n = 3), pancreas (n = 2), breast (n = 2),stomach (n = 1), esophagus (n = 2) and uterus (n = 1). All 15 patients underwent surgery following their 68Ga-FAPi-46 PET scan within a mean time interval of 16.1±14.4 days (range 1 – 50 days). For two patients the tumor was deemed unresectable. 68Ga-FAPi-46 SUVmax and IHC scores were higher in cancer tissue than in normal tissue: mean 68Ga-FAPi-46 SUVmax 7.4±4.6 (range 1.5-15.9) vs 1.6±1.2 (range 0.4-5.1), (p < 0.001) and mean FAP IHC score 2.38±0.65 vs 0.54±0.66 (p < 0.001), respectively. The FAP IHC scores strongly correlated with 68Ga-FAPi-46 SUVmax (p = 0.001, repeated measures correlation r = 0.85 (95% CI 0.53-0.95), p < 0.001). Conclusions: 68Ga-FAPi-46 PET biodistribution across multiple cancers strongly correlates with FAP tissue expression as measured by IHC. This translational validation paves the way for large scale prospective trials on the use of 68Ga-FAPi-46 PET/CT as a biomarker and stratification tool for FAP-targeted therapies. Clinical trial information: NCT04147494