Association studies are the most powerful method available for identifying modest gene effects in complex disorders, but they often produce inconsistent results. With the rapidly growing SNP databases, haplotype maps and high throughput genotyping, the use of association studies is expected to increase; therefore, it is critical and timely that the problems with study design are identified and fixed. We questioned if unrecognized allele and genotype frequency variations in controls could be responsible for some of the inconsistent association findings. We performed a population genetic study of apolipoprotein E (APOE) and cytochrome P450 2D6 (CYP2D6) in 1,748 individuals ranging in age from newborns to centenarians. Although APOE and CYP2D6 are two of the most commonly used candidate genes, this is the first study to examine age- and gender-specific frequency distributions over the entire age spectrum, using a large, ethnically and geographically uniform population. We found significant, previously unrecognized variations in APOE allele frequencies, and deviations from Hardy-Weinberg expectations in CYP2D6 genotype frequencies starting at birth. The allele frequency variations within controls were larger than some reported case-control differences. We demonstrate that unrecognized frequency fluctuations in controls are a serious and potentially common confounder whose impact on association studies has not been appreciated, and one that can be addressed with proper study design. We recommend that population genetic studies be performed on commonly used candidate markers and that rigorous standards be applied for case-control matching. © Springer-Verlag 2005.