CYP2A6 genotyping is of clinical importance – CYP2A6 gene variants influence nicotine metabolism and are associated with nicotine dependence, cigarettes per day, smoking cessation and the risk for tobacco-associated cancers. CYP2A6 gene variants also influence the metabolism of therapeutic drugs, such as the anticancer agents, tegafur and letrozole. Over the years, CYP2A6 genotyping methods have evolved to incorporate novel gene variants and to circumvent genotyping errors resulting from the high degree of homology between CYP2A6 and neighboring CYP2A genes. Herein, CYP2A6 genotyping strategies are described for commonly genotyped functionally significant alleles including SNPs, small insertions/deletions and more complex structural variants. The methods presented utilize higher throughput SYBR green real-time PCR technology in addition to standard thermocycling.