Background: The cytochrome P450 (CYP) 3A5 enzyme contributes to the metabolism of many drugs. Single nucleotide polymorphisms in the CYP3A5 gene ( CYP3A5 ⁎ 3C and CYP3A5 ⁎ 6) are associated with decreased CYP3A5 expression in the liver. We designed a multiplex genotyping assay to detect the CYP3A5 ⁎ 3C and CYP3A5 ⁎ 6 polymorphisms in a single polymerase chain reaction (PCR) and a single pyrosequencing reaction. Methods: A multiplex PCR assay was designed to simultaneously amplify 2 fragments, one containing the CYP3A5 ⁎ 3C polymorphism and the other containing the CYP3A5 ⁎ 6 polymorphism. Following PCR, multiplex genotyping was performed with pyrosequencing analysis. Results: Patient samples ( n = 69) were analyzed for the CYP3A5 ⁎ 3C and CYP3A5 ⁎ 6 polymorphisms using the multiplex PCR-pyrosequencing assay. Genotypes obtained by the multiplex reaction were in 100% concordance with genotypes obtained using simplex PCR-pyrosequencing ( n = 69) and direct DNA sequencing ( n = 29). Conclusions: The advantage of this method is that the CYP3A5 ⁎ 3C and CYP3A5 ⁎ 6 polymorphism can be amplified in a single PCR reaction and genotyped in a single pyrosequencing reaction. This combined approach improves the time-efficiency and decreases the cost of CYP3A5 genotyping.