The purpose of this exploratory study was to quantify the relationship between scalar-based measures of diffusion tensor imaging (DTI) and histologically derived microstructural measurements in precisely colocalized rat rotator cuff muscle tissue and to compare the results when imaged at 0.25- and 0.5-mm isotropic resolutions. Four Lewis rats subject to a unilateral chronic massive rotator cuff tear model were evaluated on a 3-T preclinical MRI scanner using spin echo DTI sequences at 0.25- and 0.5-mm isotropic resolutions, and histology was subsequently performed. Fractional anisotropy (FA), mean diffusivity (MD), and radial diffusivity (RD) were calculated. Whole muscle myofiber boundary segmentation was performed, and muscle fiber diameter and cross-sectional area were calculated on slides that passed rigorous histologic quality control. Scatter plots were generated on a pixel-by-pixel basis from meticulously colocalized DTI and histology data. Pearson's correlations were performed. Twenty-two distinct supraspinatus and infraspinatus muscle locations from two rats were included. Negligible correlations were found between DTI metrics, including FA, MD, and RD, and histological measurements, including muscle fiber diameters and cross-sectional areas. Using the most commonly employed spin echo DTI sequences with intermediate diffusion times, there may be negligible sensitivity to direct measures of muscle tissue microstructure. Our findings underscore the need for further research with optimized imaging parameters to enhance our knowledge regarding the capability of DTI to determine important features of muscle microstructure.