Atrial fibrillation (AF), often accompanied by atrial fibrosis, is challenging to diagnose sub-clinically and reverse once established. Molecular imaging targeting excess atrial collagen may enable earlier detection of atrial fibrosis and its associated arrhythmias. We used the collagen I-binding peptide EP-3533 and our novel 'T-peptide' targeting matrix metalloproteinases-2-digested collagen IV to image interstitial atrial fibrosis, AF and heart failure (HF) that develop in the double transgenic mouse model dnPI3K-Mst1 (termed 'AF + HF'). Ex vivo and in vivo imaging were performed using near-infrared scans and positron emission tomography (PET) with probes conjugated to Cyanine5.5 and copper-64, respectively. Both tracers significantly accumulated in fibrotic atria compared to non-transgenic controls, with specific T-peptide uptake relative to a mutated 'S-peptide'. Pharmacokinetic profiling demonstrated good tracer plasma stability and fast renal clearance. These results highlight the potential of collagen-targeted peptide tracers, particularly the disease stage-sensitive T-peptide, to improve diagnosis and monitoring of atrial fibrosis and AF.