Hypothesis: – Congenital unilateral conductive hearing impairment alters auditory white matter tract connections of the brain. Background: – Unilateral conductive hearing loss has negative effects on speech perception in noise and sound localization ability. This study examines auditory white matter tracts in a rat model with surgically induced unilateral ear canal atresia, compared with controls. Materials and methods: – Twelve-month euthanized rats, 7 with left-sided surgically created ear canal atresia (UCA) and 4 controls, were scanned in a 9.4 T MRI scanner for animal research purposes. Diffusion-weighted images were acquired. Two different methods for probabilistic fiber tracking were used. Fractional anisotropy (FA) was calculated from whole brains and from tracts between defined auditory regions of interest. Results: – Both methods of fiber tracking show significantly higher global FA in rats with UCA. Within-group comparisons in UCA show higher FA on the hearing side between the cochlear nucleus and inferior colliculus, compared with the side of hearing loss, while in controls, there was no asymmetry. Lower FA was also found between the medial geniculate complex and the auditory cortex on the left side than on the right in UCA, in contrast to controls, where there was no difference between the hemispheres. Conclusion: – The asymmetry found in UCA indicates alterations of auditory white matter tracts as a response to asymmetric hearing, partly confirming previous findings in experiments on ferrets.