This study used in vivo magnetic resonance imaging (MRI) to identify age dependent brain structural characteristics in Dunkin Hartley guinea pigs. Anatomical T2-weighted images, diffusion kurtosis (DKI) imaging, and T2 relaxometry measures were acquired from a cohort of male guinea pigs from postnatal day (PND) 18–25 (juvenile) to PND 46–51 (adolescent) and PND 118–123 (young adult). Whole-brain diffusion measures revealed the distinct effects of maturation on the microstructural complexity of the male guinea pig brain. Specifically, fractional anisotropy (FA), as well as mean, axial, and radial kurtosis in the corpus callosum, amygdala, dorsal-ventral striatum, and thalamus significantly increased from PND 18–25 to PND 118–123. Age-related alterations in DKI measures within these brain regions paralleled the overall alterations observed in the whole brain. Age-related changes in FA and kurtosis in the gray matter-dominant parietal cerebral cortex and dorsal hippocampus were less pronounced than in the other brain regions. The regional data analysis revealed that between-age changes of diffusion kurtosis metrics were more pronounced than those observed in diffusion tensor metrics. The age-related anatomical differences reported here may be important determinants of the age-dependent neurobehavior of guinea pigs in different tasks.