Background Recent large-scale Genome-Wide Association Studies (GWAS) have identified genetic loci associated with various substance use traits. However, little is known about the functional mechanisms through which these genetic loci ascertain their effect. This study aims to identify and functionally annotate genetic loci associated with substance use traits based on their role in genetic regulation of gene expression. Methods We evaluated expression quantitative trait loci (eQTLs) in human post mortem tissue from 10 brain regions and whole blood of the Genotype-Tissue Expression (GTEx) database. The role of eQTLs was examined for eight substance use traits, using summary statistics from GWAS of: alcohol consumption (N=70,460), cigarettes per day (CPD; N=38,181), former vs. current smoker (N=41,278), age of smoking initiation (N=24,114), ever smoker (N= 74,035), cocaine dependence (N=4,769), cannabis dependence (N=12,168), and lifetime cannabis use (N=32,330). Results eQTLs with significant trait association (FDR <0.05) were found for alcohol consumption, CPD, former vs. current smoker, cocaine dependence, and cannabis dependence in various tissues. The eGenes targeted by these significant eQTLs often differed from their nearest genes, indicating that proximity alone is a limited measure to determine the causal gene. Using this methodology, we elucidated eQTLs and eGenes which were not picked up by the original GWAS, and were able to determine through which tissue these loci exert their effects. Discussion Annotating genes based on transcriptomic regulation in brain and non-brain tissues improves the identification of novel loci and the functional characterization of genetic risk loci for substance use traits.