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Approximately half of HIV-infected people exhibit cognitive impairment, which has been related to cerebral white matter damage. Despite the effectiveness of antiretroviral (ARV) treatment, cognitive impairment remains common even in individuals with undetectable viral loads. One explanation for this may be subtherapeutic concentrations of some ARVs in the CNS. We utilized diffusion tensor imaging (DTI) to investigate the relationship of white matter integrity to cognitive impairment and ARV treatment variables, including CSF viral load and an index of the CNS penetration of ARVs. Participants included 39 HIV-infected (HIV+) individuals and 25 seronegative subjects. DTI indices were mapped onto a common whole-brain white matter tract skeleton, allowing between-subject voxelwise comparisons. The total HIV+ group exhibited abnormal white matter in the internal capsule, inferior longitudinal fasciculus, and optic radiation; while HIV+ with AIDS exhibited more widespread damage, including in the internal capsule and the corpus callosum. Cognitive impairment in HIV+ was related to white matter injury in the internal capsule, corpus callosum, and superior longitudinal fasciculus. White matter injury was not found to be associated with HIV viral load or estimated CNS penetration of ARVs. DTI was useful in identifying changes in white matter tracts associated with more advanced HIV infection. Relationships between diffusion alterations in specific white matter tracts and cognitive impairment support the potential utility of DTI in examining the anatomical underpinnings of HIV-related cognitive impairment. The study also confirms that CNS injury is evident in persons infected with HIV despite effective ARV treatment