One of the fundamental unmet clinical needs within cardiac electrophysiology is intraoperative assessment of catheter ablation, which can lead to recurrent arrhythmias and subsequent complications if ineffective. This work demonstrates photoacoustic imaging (PAI) of radiofrequency ablation (RFA) lesions in an in vivo swine model (n=3). Spectral unmixing of PAI data provides local myocardial characterization (e.g., oxygen saturation & tissue ablation) by overlaying unmixed PAI images with B-mode ultrasound imaging (PAI/US), with the latter providing anatomical context. Based on stained gross pathology, areas of central tissue necrosis coincided with increases in unmixed ablated regions of the myocardium. An average contrast-to-noise ratio of 2.8±0.2 confirmed lesion detectability, while the lesion dimensions quantified from PAI and pathology did not present significant differences. In vivo PAI of RFA lesions to determine ablation characteristics could lead to a paradigm shift in catheter ablation assessment and improve clinical outcomes.