Background: Letrozole is a non-steroidal aromatase inhibitor (AI) used to treat hormone receptor positive (HR+) breast cancer. Variability in letrozole efficacy and toxicity may be partially attributable to variable systemic drug exposure, which may be influenced by germline variants in the enzymes responsible for letrozole metabolism, including CYP2A6. The objective of this genome-wide association study (GWAS) was to identify genetic variants that affect steady state letrozole concentrations. Methods: The Exemestane and Letrozole Pharmacogenetics (ELPh) Study randomized 503 post-menopausal patients with HR+ non-metastatic breast cancer to exemestane or letrozole treatment. Germline DNA was collected pre-treatment and blood samples were collected after 1 or 3 months of treatment to measure steady-state letrozole concentration via HPLC/MS. Genome-wide genotyping was conducted on the Infinium Global Screening Array to the Haplotype Reference Panel ( > 2 million variants). The association of each polymorphism with square-root transformed letrozole concentration was tested in self-reported white patients via linear regression using the standard alpha for genome-wide significance (α = 5x10⁻⁸) assuming an additive genetic model and correcting for age and body mass index. Results: 228 patients met inclusion criteria and had all necessary data. Each variant allele of rs7937 a patient carried increased their letrozole concentration ~22.9 ng/mL (standard error = 4.01, p = 3.51x10⁻⁸, Table) and this variant explained 13% of the variability in letrozole concentrations. rs7937 is located ~50 kB upstream of CYP2A6, and has previously been identified in GWAS of CYP2A6-related phenotypes, including nicotine metabolism and lung cancer. Conclusions: This GWAS confirmed that steady-state letrozole concentrations are partially determined by germline polymorphisms affecting CYP2A6 activity. If letrozole concentrations affect treatment efficacy or toxicity, CYP2A6 genetics may be useful to individualize letrozole dosing to improve clinical outcomes in patients with HR+ breast cancer.