This study was aimed at developing a protocol for an integrated energetic and functional characterization of transgenic mouse models with cardiomyopathies. Using acquisition-weighted 2D 31P chemical shift imaging at a field strength of 9.4 Tesla, we were able to acquire 31P MR spectra of good quality from the entire thorax of the mouse with high spatial resolution at defined regions of the heart (anterior, lateral, posterior, and septal wall) within a reasonable acquisition time of about 75 min. 2D data sets were quantified after application of appropriate correction factors (blood + partial saturation) using a self-developed software module in direct correlation to the morphological 1H MR image. Analysis of a transgenic cardiomyopathy model (double mutant: cardiospecific iNOS overexpression and lack of myoglobin (TGiNOS/myo-/-) revealed that cardiac dysfunction (EF: 53.9±3.8%) was associated with an impaired energy homeostasis (PCr/ATP 1.54±0.18) over the entire left ventricle (WT: EF 69.7±3.5%, PCr/ATP 2.06±0.22, n=5, P<0.05). The spectroscopic data acquired in vivo were validated against 31P MR spectra of perchloric acid extracts from the same hearts (PCr/ATP: 1.87±0.21 (WT), 1.39±0.17 (TGiNOS/myo-/-), n=5, P<0.05). The presented method allows the noninvasive, repetitive measurement of cardiac anatomy and function together with the regional energy state in transgenic mouse models in one experiment.