Background: Obesity is the most common comorbidity and risk factor for heart failure (HF) in patients with preserved ejection fraction (HFpEF), a subclass containing more than 50% of HF cases. However, effective treatment of HFpEF remains scarce due to the incomplete understanding of the mechanisms. Methods: A mouse line (C57/B6J) of Floxed-stop Overexpression of IF1 (FEI) was generated and maintained for Cre-mediated conditional IF1 overexpression. We observed that severe obesity became a fixed phenotype in inbred FEI mice after inbreeding for 5-6 generations. Targeted-Locus-Amplification (TLA) analyses revealed that FEI inbreeding triggered a genomic rearrangement in a non-coding region of chromosome 1. A heterozygous line with genotype and phenotype fixation was obtained via outbreeding with wild type mice. In the present study, we focus on assessing the cardiac phenotype of FEI mice using echocardiography, histology, and exercise tolerance tests. Results: The early onset of obesity was consistent in the heterozygotes of obese FEI mice with increased peripheral fats and circulating tissue lipids, as well as reduced systemic metabolic rates. FEI mice also exhibited increased food intake, fasting blood glucose, insulin, and leptin with impaired glucose/insulin tolerance. Echocardiography revealed that the FEI mice developed concentric left ventricular hypertrophy with preserved ejection fraction. However, E/A ratio results from Doppler flow measurement at mitral valve suggest diastolic dysfunction in the FEI mice. Heart weight to tibial length ratio was significantly increased in FEI compared with WT mice. H&E and Trichrome blue staining on heart sections exhibited increased cardiomyocyte hypertrophy and cardiac fibrosis. Treadmill exercise tolerance test revealed a significantly decreased exercise tolerance in FEI mice. Conclusions: We conclude that the cardiac phenotypes in the obese FEI mice closely recapitulate obese patients who suffer from HFpEF. Thus, further studies on these mice should yield novel and translational insight into the molecular and metabolic mechanisms underlying the HFpEF development, leading to improved treatments.