AbstractThis study was designed to investigate the potential differences between Spaniards and Ecuadorian Mestizo people regardingCYP2A6*1A,CYP2A6*1B1,CYP2A6*1x2A,CYP2A6*9A, andCYP2A6*4Avariant alleles at theCYP2A6gene and also to compare the observed frequencies with those previously reported in different ethnic groups. DNA from 234 Spaniard and 300 Ecuadorian subjects were analyzed by either PCR or PCR‐restriction fragment length polymorphism. Differences between Spaniards and Mestizo Ecuadorians were detected in relation to the frequencies of the alleles linked to either absent enzyme activity,CYP2A6*4A(4 and 7.1%, respectively), or reduced CYP2A6 enzyme activity,CYP2A6*9A(6.4 and 10.3%, respectively).CYP2A6*4AandCYP2A6*9Afrequencies in Ecuadorians were higher than those in Africans or Caucasian groups and lower than those in Asians. This study provides, for the first time, the result of the analysis ofCYP2A6allele frequency in a South American population and demonstrates the presence of ethnic differences inCYP2A6genetic variants between Spaniards and Mestizo Ecuadorians, which should be considered in allele–disease association studies and, in particular, in those involvingCYP2A6genetic polymorphisms and tobacco‐related cancer.