Polyadenylation of nascent transcripts is an essential step for most mRNAs in eukaryotic cells. It is directly involved in the termination of transcription and is coupled with other steps of pre-mRNA processing. Recent studies have shown that transcript variants resulting from alternative polyadenylation are widespread for human and mouse genes, contributing to the complexity of mRNA pool in the cell. In addition to 3′-most exons, alternative polyadenylation sites (or poly(A) sites) can be located in internal exons and introns. Identification of poly(A) sites in genomes is critical for understanding the occurrence and significance of alternative polyadenylation events. Bioinformatic methods using cDNA sequences, Expressed Sequence Tags (ESTs), and Trace offer a sensitive and systematic approach to detect poly(A) sites in genomes. Various criteria can be employed to enhance the specificity of the detection, including identifying sequences derived from internal priming of mRNA and polyadenylated RNAs during degradation.