Background Structural and functional MRI studies have revealed brain alterations associated with opioid use disorder (OUD). However, previous research has been limited by small sample sizes, few female participants, and single-modality analyses. Purpose To identify structural and functional brain alterations in individuals with OUD using whole-brain data-driven methods to analyze T1-weighted MRI and resting-state functional MRI (rsfMRI). Materials and Methods This secondary analysis of the Collaboration Linking Opioid Use Disorder and Sleep study compared participants with OUD (data collection February 2021 to February 2023) receiving methadone treatment with healthy control participants (data collection February 2018 to May 2023). T1-weighted MRI and rsfMRI were analyzed using tensor-based morphometry and intrinsic connectivity distribution, respectively. Primary outcome measures included regional brain volumes and functional connectivity. Voxel-wise linear regression was used to assess group differences, with family-wise error correction. Pearson partial correlations were used to examine structure-function relationships. Results Compared with healthy control participants (n = 105; median age, 27 years [IQR, 23-37 years]; 58 female), participants with OUD (n = 103; median age, 37 years [IQR, 31.5-46 years]; 62 male) showed smaller volumes (corrected P < .05) in the thalamus (β = -17.42 [95% CI: -26.56, -8.27]) and right medial temporal lobe (β = -8.02 [95% CI: -12.25, -3.78]). Larger volumes (corrected P < .05) were seen in the brainstem (pons and medulla, β = 15.21 [95% CI: 7.03, 23.40]; midbrain, β = 13.04 [95% CI: 6.61, 19.47]) and cerebellum (right, β = 14.96 [95% CI: 7.25, 22.67]; left, β = 14.88 [95% CI: 7.32, 22.43]). An interaction between sex and group was found for medial prefrontal cortex volume (β = -19.38, corrected P < .05), with female participants having smaller volumes than male participants in the OUD group. Increased functional connectivity (corrected P < .05) was noted in the thalamus (β = 0.50 [95% CI: 0.25, 0.75]), right medial temporal lobe (β = 0.43 [95% CI: 0.21, 0.66]), right cerebellum (β = 0.46 [95% CI: 0.21, 0.71]), and brainstem (β = 0.48 [95% CI: 0.22, 0.74]) in the OUD group. Structure and function were positively correlated in the cerebellum (Pearson r = 0.32 [95% CI: 0.17, 0.44], P < .001) and brainstem (Pearson r = 0.23 [95% CI: 0.09, 0.37], P = .002). Conclusion Individuals with OUD showed overlapping structural and functional brain alterations in opioid receptor-dense regions compared with healthy control participants. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Filippi and Messina in this issue.