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Abstract: Background and aims: Disentangling the putative impact of cannabis on brain morphology from other comorbid substance use is critical. After controlling for the effects of nicotine, alcohol and multi‐substance use, this study aimed to determine whether frequent cannabis use is associated with significantly smaller subcortical grey matter volumes. Design: Exploratory analyses using mixed linear models, one per region of interest (ROI), were performed whereby individual differences in volume (outcome) at seven subcortical ROIs were regressed onto cannabis and comorbid substance use (predictors). Setting: Two large population‐based twin samples from the United States and Australia. Participants: A total of 622 young Australian adults [66% female; μage = 25.9, standard deviation SD) = 3.6] and 474 middle‐aged US males (μage = 56.1SD = 2.6) of predominately Anglo‐Saxon ancestry with complete substance use and imaging data. Subjects with a history of stroke or traumatic brain injury were excluded. Measurements: Magnetic resonance imaging (MRI) and volumetric segmentation methods were used to estimate volume in seven subcortical ROIs: thalamus, caudate nucleus, putamen, pallidum, hippocampus, amygdala and nucleus accumbens. Substance use measurements included maximum nicotine and alcohol use, total life‐time multi‐substance use, maximum cannabis use in the young adults and regular cannabis use in the middle‐aged males. Findings: After correcting for multiple testing (P = 0.007), cannabis use was unrelated to any subcortical ROI. However, maximum nicotine use was associated with significantly smaller thalamus volumes in middle‐aged males. Conclusions: In exploratory analyses based on young adult and middle‐aged samples, normal variation in cannabis use is unrelated statistically to individual differences in brain morphology as measured by subcortical volume. [ABSTRACT FROM AUTHOR]