We have developed the population tube (poptube) system for sensitive detection and large-scale sampling of DNA sequence variation in several human populations of wide geographic distribution. In this methodology, genomic DNAs from five individuals in a population are PCR amplified en masse to maximize deliberately the chances of forming heteroduplexes among allelic variants. Interpopulation mixing is performed in a separate set of tubes containing one individual from each of five populations as well as a reference chimpanzee sample deliberately chosen to be different from all humans. Mismatches at sites of allelic variation retard the electrophoresis and reduce the stability of heteroduplex molecules. The products are electrophoresed on denaturing gradient gels where detection of heteroduplexes is accomplished readily. Using poptubes, we have discovered a rare variant in an otherwise highly conserved 440-bp segment in the long intron of the glucose-6-phosphate dehydrogenase (C6PD) gene. The polymorphism at this X-chromosome locus could be only detected in males by mixing samples, as homoduplexes for both alleles co-focus on denaturing gradient electrophoresis.