The development of high‐throughput DNA sequencing techniques has made direct DNA sequencing of PCR‐amplified genomic DNA a rapid and economical approach to the identification of polymorphisms that may play a role in disease. Point mutations as well as small insertions or deletions are readily identified by DNA sequencing. The mutations may be heterozygous (occurring in one allele while the other allele retains the normal sequence) or homozygous (occurring in both alleles). Sequencing alone cannot discriminate between true homozygosity and apparent homozygosity due to the loss of one allele due to a large deletion. In this unit, strategies are presented for using PCR amplification and automated fluorescence‐based sequencing to identify sequence variation. The size of the project and laboratory preference and experience will dictate how the data is managed and which software tools are used for analysis. A high‐throughput protocol is given that has been used to search for mutations in over 200 different genes at the Harvard Medical School – Partners Center for Genetics and Genomics (HPCGG, http://www.hpcgg.org/). Curr. Protoc. Hum. Genet. 57:7.9.1‐7.9.31. © 2008 by John Wiley & Sons, Inc.