Osmotic minipumps containing methadone or saline were implanted in pregnant rats on E16. Pups were born and underwent ex vivo diffusion tensor imaging on postnatal day 21 (P21). Compared to sham, methadone pups have abnormal directional diffusion (top panel) and reduced fractional anisotropy (FA, A and B) in major white matter tracts. Notably, axial diffusivity (AD) is significantly decreased in the corpus callosum of methadone pups (C), while radial diffusivity (RD) remained unchanged (D). While AD in the external capsule was not statistically different, RD was increased in methadone pups compared to shams. These data are consistent with reduced integrity of white matter microstructure, axon and myelin injury. *p<0.05 n=4–5/group.