We aimed to use diffusion tensor imaging (DTI) to detect alterations in white matter microstructure in older patients with breast cancer receiving chemotherapy. Methods: We recruited women age ≥60 years with stage I-III breast cancer (chemotherapy [CT] group; n = 19) to undergo two study assessments: at baseline and within one month after chemotherapy. Each assessment consisted of a brain magnetic resonance imaging scan with DTI and neuropsychological (NP) testing using the National Institutes of Health (NIH) Toolbox Cognition Battery. An age-and sex-matched group of healthy controls (HC, n = 14) underwent the same assessments at matched intervals. Four DTI parameters (fractional anisotropy [FA], mean diffusivity [MD], axial diffusivity [AD], and radial diffusivity [RD]) were calculated and correlated with NP testing scores. Results: For CT group but not HCs, we detected statistically significant increases in MD and RD in the genu of the corpus callosum from time point 1 to time point 2 at p b 0.01, effect size:0.3655 and 0.3173, and 95% confidence interval: from 0.1490 to 0.5821, and from 0.1554 to 0.4792, for MD and RD respectively. AD values increased for the CT group and decreased for the HC group over time, resulting in significant between-group differences (p = 0.0056, effect size:1.0215, 95% confidence interval: from 0.2773 to 1.7657). There were no significant correlations between DTI parameters and NP scores (p N 0.05). Conclusions: We identified alterations in white matter microstructures in older women with breast cancer undergoing chemotherapy. These findings may potentially serve as neuroimaging biomarkers for identifying cognitive impairment in older adults with cancer.