Background: Previous studies have reported white matter (WM) brain alterations in asymptomatic patients with human immunodeficiency virus (HIV). Methods: We compared diffusion tensor imaging (DTI) derived WM fractional anisotropy (FA) between HIV-patients with and without mild macroscopic brain lesions determined using standard magnetic resonance imaging (MRI). We furthermore investigated whether WM alterations co-occurred with neurocognitive deficits and depression. We performed structural MRI and DTI for 19 patients and 19 age-matched healthy controls. Regionally-specific WM integrity was investigated using voxel-based statistics of whole-brain FA maps and region-of-interest analysis. Each patient underwent laboratory and neuropsychological tests. Results: Structural MRI revealed no lesions in twelve (HIV-MRN) and unspecific mild macrostructural lesions in seven patients (HIV-MRL). Both analyses revealed widespread FA-alterations in all patients. Patients with HIV-MRL had FA-alterations primarily adjacent to the observed lesions and, whilst reduced in extent, patients with HIV-MRN also exhibited FA-alterations in similar regions. Patients with evidence of depression showed FA-increase in the ventral tegmental area, pallidum and nucleus accumbens in both hemispheres, and patients with evidence of HIV-associated neurocognitive disorder showed widespread FA-reduction. Conclusion: These results show that patients with HIV-MRN have evidence of FA-alterations in similar regions that are lesioned in HIV-MRL patients, suggesting common neuropathological processes. Furthermore, they suggest a biological rather than a reactive origin of depression in HIV-patients.