Purpose <p>Myocardial infarction (MI) triggers complex cellular responses essential for tissue repair and remodeling, including myofibroblast activation. Fibroblast activation protein alpha (FAP) identifies activated myofibroblasts post-MI, however its spatial distribution relative to the scar and area at risk (AAR) is unclear. Non-invasive FAP-imaging with PET radiotracer ⁶⁸&#xa0;Ga-FAPI-46 shows uptake beyond the infarct scar. We therefore aimed to characterize FAP expression in the AAR using a myocardial ischemia–reperfusion (MI/R) model in mice.</p> Procedures <p>We induced MI/R in male C57BL/6N mice. The AAR was identified by <i>in vivo</i> lectin staining, and expression of FAP, CD68, and hypoxic tissues were measured using immunohistochemistry. Spatial FAP was further interrogated by ⁶⁸&#xa0;Ga-FAPI-46 in mice by autoradiography and humans by PET. Additionally, human cardiac tissues from acute MI patients were examined for fibroblasts and inflammatory cells by expression of FAP, CD13, and α-smooth muscle actin.</p> Results <p>FAP expression peaked three days post-MI/R predominantly within the AAR (p &lt; 0.05 vs. d0). Consistent between murine models and human tissues, FAP⁺ myofibroblasts accumulated within the infarct scar and borderzone, occasionally extending into non-ischemic myocardium. CD68⁺ macrophages peaked similarly at three days post-MI/R (p &lt; 0.05 vs. d0). FAP expression weakly correlated with CD68 but not with extent of ischemic or hypoxic territory post-MI/R. FAP imaging in mice and humans revealed aligned non-uniform ⁶⁸&#xa0;Ga-FAPI-46 uptake extending from the infarct scar into surviving myocardium after MI.</p> Conclusions <p>Our findings demonstrate a distinct FAP expression pattern post-MI/R. The alignment of <i>ex vivo</i> ⁶⁸&#xa0;Ga-FAPI-46 signal with myofibroblasts in the AAR supports its identification of a unique substrate in myocardial injury complementing other non-invasive imaging measurements of perfusion, viability and fibrosis.</p>