Sequence annotation is essential for genomics-based research. Investigators of a specific genomic region who have developed abundant local discoveries such as genes and genetic markers, or have collected annotations from multiple resources, can be overwhelmed by the difficulty in creating local annotation and the complexity of integrating all the annotations. Presenting such integrated data in a form suitable for data mining and high-throughput experimental design is even more daunting. DNannotator, a web application, was designed to perform batch annotation on a sizeable genomic region. It takes annotation source data, such as SNPs, genes, primers, and so on, prepared by the end-user and/or a specified target of genomic DNA, and performs de novo annotation. DNannotator can also robustly migrate existing annotations in GenBank format from one sequence to another. Annotation results are provided in GenBank format and in tab-delimited text, which can be imported and managed in a database or spreadsheet and combined with existing annotation as desired. Graphic viewers, such as Genome Browser or Artemis, can display the annotation results. Reference data (reports on the process) facilitating the user's evaluation of annotation quality are optionally provided. DNannotator can be accessed at http://sky.bsd.uchicago.edu/DNannotator.htm.