We characterized the microstructural response of the myocardium to cardiovascular disease using diffusion tensor imaging (DTI) and performed histological validation by intact, un-sectioned, three-dimensional (3D) histology using a tissue-clearing technique. The approach was validated in normal (n = 7) and ischemic (n = 8) heart failure model mice. Whole heart fiber tracking using DTI in fixed ex-vivo mouse hearts was performed, and the hearts were processed with the tissue-clearing technique. Cardiomyocytes orientation was quantified on both DTI and 3D histology. Helix angle (HA) and global HA transmurality (HAT) were calculated, and the DTI findings were confirmed with 3D histology. Global HAT was significantly reduced in the ischemic group (DTI: 0.79 ± 0.13°/% transmural depth [TD] and 3D histology: 0.84 ± 0.26°/%TD) compared with controls (DTI: 1.31 ± 0.20°/%TD and 3D histology: 1.36 ± 0.27°/%TD, all p < 0.001). On direct comparison of DTI with 3D histology for the quantitative assessment of cardiomyocytes orientation, significant correlations were observed in both per-sample (R 2 = 0.803) and per-segment analyses (R 2 = 0.872). We demonstrated the capability and accuracy of DTI for mapping cardiomyocytes orientation by comparison with the intact 3D histology acquired by tissue-clearing technique. DTI is a promising tool for the noninvasive characterization of cardiomyocytes architecture.