Insertional mutagenesis is a powerful method for gene discovery. To identify the location of insertion sites in the genome linker based polymerase chain reaction (PCR) methods (such as splinkerette-PCR) may be employed. We have developed a web application called iMapper (Insertional Mutagenesis Mapping and Analysis Tool) for the efficient analysis of insertion site sequence reads against vertebrate and invertebrate Ensembl genomes. Taking linker based sequences as input, iMapper scans and trims the sequence to remove the linker and sequences derived from the insertional mutagen. The software then identifies and removes contaminating sequences derived from chimeric genomic fragments, vector or the transposon concatamer and then presents the clipped sequence reads to a sequence mapping server which aligns them to an Ensembl genome. Insertion sites can then be navigated in Ensembl in the context of genomic features such as gene structures. iMapper also generates test-based format for nucleic acid or protein sequences (FASTA) and generic file format (GFF) files of the clipped sequence reads and provides a graphical overview of the mapped insertion sites against a karyotype. iMapper is designed for high-throughput applications and can efficiently process thousands of DNA sequence reads.