Pharmacogenetic testing for CYP3A4 is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the CYP3A4 variants included in clinical tests. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 30 DNA samples derived from Coriell cell lines for CYP3A4. Samples were distributed to five volunteer laboratories for genotyping using a variety of commercially available and laboratory-developed tests. Sanger and next-generation sequencing were also utilized by some of the laboratories. Whole-genome sequencing data from the 1000 Genomes Projects were utilized to inform genotype. Twenty CYP3A4 alleles were identified in the 30 samples characterized for CYP3A4: CYP3A4∗4, ∗5, ∗6, ∗7, ∗8, ∗9, ∗10, ∗11, ∗12, ∗15, ∗16, ∗18, ∗19, ∗20, ∗21, ∗22, ∗23, ∗24, ∗35, and a novel allele, CYP3A4∗38. Nineteen additional samples with preexisting data for CYP3A4 or CYP3A5 were re-analyzed to generate comprehensive reference material panels for these genes. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.