Biomolecule sequences and structures of land, air and water species are determined rapidly and the data entries are unevenly distributed for different organisms. It frequently leads to the BLAST results of homologous search containing undesirable entries from organisms living in different environments. To reduce irrelevant searching results, a separate database for comparative genomics is urgently required. A comprehensive bioinformatics tool set and an integrated database, named Bioinformatics tools for Marine and Freshwater Genomics (BiMFG), are constructed for comparative analyses among model species and underwater species. Novel matching techniques based on conserved motifs and/or secondary structure elements are designed for efficiently and effectively retrieving and aligning remote sequences through cross-species comparisons. It is especially helpful when sequences under analysis possess low similarities and unresolved structural information. In addition, the system provides core techniques of multiple sequence alignment, multiple second structure profile alignment and iteratively refined multiple structural alignments for biodiversity analysis and verification in marine and freshwater biology. The BiMFG web server is freely available for use at http://bimfg.cs.ntou.edu.tw/.