Objective: Major depressive disorder (MDD) occurs frequently in adolescents, but the neurobiology of depression in youth is poorly understood. Structural neuroimaging studies in both adult and pediatric populations have implicated frontolimbic neural networks in the pathophysiology of MDD. Diffusion tensor imaging (DTI), which measures white matter (WM) microstructure, is a promising tool for examining neural connections and how they may be abnormal in MDD. Method: We used two separate approaches to analyze DTI data in adolescents with MDD (n = 14) compared with healthy volunteers (n = 14). Results: The first, hypothesis-driven approach was to use probabilistic tractography to delineate tracts arising from the subgenual anterior cingulate cortex (ACC). Adolescents with MDD demonstrated lower fractional anisotropy (FA) in the WM tract connecting subgenual ACC to amygdala in the right hemisphere. The second, exploratory approach was to conduct a voxelwise comparison of FA. This analysis revealed 10 clusters where adolescents with MDD had significantly lower (uncorrected) FA than the healthy group within WM tracts including right and left uncinate and supragenual cingulum. Conclusions: These preliminary data support the hypothesis that altered WM microstructure in frontolimbic neural pathways may contribute to the pathophysiology of MDD in adolescents. (Contains 3 figures and 3 tables.)