Here we present the first large-scale effort at genotyping using a novel sequencing method, Pyrosequencing™, as a method for genotyping of single nucleotide polymorphisms (SNPs). Pyrosequencing™ genotypes were validated through duplicate analysis of 1,022 genotypes using the PSQ96™ instrument for pyrosequencing and TaqMan® for 5′nuclease assays. Identical results were obtained using both methods. In a small pilot study, a pooling strategy using Pyrosequencing™ was successfully tested. We conclude that Pyrosequencing™ is highly efficient and accurate in the analysis of SNPs and represents a promising solution to high-throughput genotyping of large sample populations. Hum Mutat 19:395–401, 2002. © 2002 Wiley-Liss, Inc.