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Expressed sequence tags (ESTs) are fragments of mRNA sequences derived through single sequencing reactions performed on randomly selected clones from cDNA libraries. To date, over 45 million ESTs have been generated from over 1400 different species of eukaryotes. For the most part, EST projects are used to either complement existing genome projects or serve as low-cost alternatives for purposes of gene discovery. However, with improvements in accuracy and coverage, they are beginning to find application in fields such as phylogenetics, transcript profiling and proteomics. This volume provides practical details on the generation and analysis of ESTs. Chapters are presented which cover creation of cDNA libraries; generation and processing of sequence data; bioinformatics analysis of ESTs; and their application to phylogenetics and transcript profiling.