Background: Controversy exists regarding the impact of CYP2D6 genotype on tamoxifen responsiveness. We examined loss of heterozygosity (LOH) at the CYP2D6 locus and determined its impact on genotyping error when tumor tissue is used as a DNA source. Methods: Genomic tumor data from the adjuvant and metastatic settings (The Cancer Genome Atlas [TCGA] and Foundation Medicine [FM]) were analyzed to characterize the impact of CYP2D6 copy number alterations (CNAs) and LOH on Hardy Weinberg equilibrium (HWE). Additionally, we analyzed CYP2D6 ∗4 genotype from formalin-fixed paraffin-embedded (FFPE) tumor blocks containing nonmalignant tissue and buccal (germline) samples from patients on the North Central Cancer Treatment Group (NCCTG) 89-30-52 tamoxifen trial. All statistical tests were two-sided. Results: In TCGA samples (n =627), the CYP2D6 LOH rate was similar in estrogen receptor (ER)-positive (41.2%) and ER-negative (35.2%) but lower in HER2-positive tumors (15.1%) (P <. 001). In FM ER+ samples (n = 290), similar LOH rates were observed (40.8%). In 190 NCCTG samples, the agreement between CYP2D6 genotypes derived from FFPE tumors and FFPE tumors containing nonmalignant tissue was moderate (weighted Kappa = 0.74; 95% CI = 0.63 to 0.84). Comparing CYP2D6 genotypes derived from buccal cells to FFPE tumor DNA, CYP2D6∗4 genotype was discordant in six of 31(19.4%). In contrast, there was no disagreement between CYP2D6 genotypes derived from buccal cells with FFPE tumors containing nonmalignant tissue. Conclusions: LOH at the CYP2D6 locus is common in breast cancer, resulting in potential misclassification of germline CYP2D6 genotypes. Tumor DNA should not be used to determine germline CYP2D6 genotype without sensitive techniques to detect low frequency alleles and quality control procedures appropriate for somatic DNA.