Background: Genetic influences underpinning complex traits are thought to involve multiple quantitative trait loci (QTLs) of small effect size. Detection of such QTL associations requires systematic screening of large numbers of DNA markers within large sample populations. Using pooled DNA on SNP microarrays to screen for allelic frequency differences between groups such as cases and controls ( called SNP Microarray and Pooling, or SNP-MaP) has been validated as an efficient solution on both 10 k and 100 k platforms. We demonstrate that this approach can be effectively applied to the truly genomewide Affymetrix GeneChip (R) Mapping 500 K Array. Results: In comparisons between five independent DNA pools (N similar to 200 per pool) on separate Affymetrix GeneChip (R) Mapping 500 K Array sets, we show that, for SNPs with minor allele frequencies > 0.05, the reliability of the rank order of estimated allele frequencies, assessed as the average correlation between allele frequency estimates across the DNA pools, was 0.948 ( average mean difference across the five pools = 0.069). Similarly, validity of the SNP-MaP approach was demonstrated by a rank-order correlation of 0.937 (average mean difference = 0.095) between the average DNA pool allele frequency estimates and the allele frequencies of an independent ( CEPH) sample of 60 unrelated individually genotyped subjects. Conclusion: We conclude that SNP-MaP can be extended for use on the Affymetrix GeneChip (R) Mapping 500 K Array, providing a cost-effective, reliable and valid initial screen of 500 K SNP microarrays in genomewide association scans.