CYP2E1, CYP2A6 and CYP3A5 enzymes belong to phase I group of drug-metabolizing enzymes, which are involved in the metabolism of various compounds and xenobiotics. Presence of polymorphisms in the genes coding for these enzymes results in interindividual variations in drug metabolism, therapeutic response and susceptibility towards various diseases. The frequencies of these variants in genes differ considerably between ethnic groups. This study was carried out to estimate the allele and genotype frequencies of common variants in CYP2E1, CYP2A6 and CYP3A5 in South Indian population. Six hundred and fifty-two unrelated healthy volunteers of South Indian origin (Andhra Pradesh, Karnataka, Kerala and Tamil Nadu) were included in this study. Polymerase chain reaction-restriction fragment length polymorphism, allele-specific PCR, real-time PCR, SNaPshot and gene sequencing methods were used for the identification of gene polymorphisms. The frequencies of CYP2E1*1B, CYP2E1*5B and CYP2E1*6 alleles in South Indian population were 14.3, 1.3 and 22.4%, respectively. The frequencies of CYP2A6*2, CYP2A6*4A and CYP2A6*5 alleles were found to be 1, 8.9 and 0.7%, respectively. The distribution of CYP3A5*3 allele was 63.5%. There were no variant alleles of CYP3A5*2, CYP3A5*4 and CYP3A5*6 in South Indian population. The frequencies of CYP2E1, CYP2A6 and CYP3A5 in the South Indian population are distinct from Caucasians, Chinese, Japanese, African Americans and other compared populations. This is the first study conducted in the South Indian population with a larger sample size. The findings of our study provide the basic genetic information for further pharmacogenomic investigations in the population.