The olive flounder Paralichthys olivaceus, is one of the most economically important marine aquaculture species, with a selective breeding program to increase aquaculture production in Korea. However, a paucity of information is available regarding the olive flounder genome. Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two BAC libraries, with large, high quality inserts and deep coverage, for P. olivaceus as a crucial part of the olive flounder genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The two libraries contain a total of 66,816 BAC clones arrayed in 174 384-well microtiter plates and correspond to 14.18 haploid genome equivalents based on olive flounder genome size of 550Mb. A random sampling of 335 BACs indicated an average insert length of 118 kb with a range of 80 to 180 kb, and 1.5% of the BACs do not contain inserts. The combined libraries have a greater than 99% probability of containing any single-copy sequence. The BAC library has been arranged in three-dimentional pools allowing screening with various PCR-based markers. BAC colonies were screened with PCR-generated gene-specific probes for the DMRT1 and 2 positive clones covering this gene fully were identified. We generated a total of 26,824 high-quality BAC-end sequences (BES), 96.5% of which were paired-BES. The average read length was 676 bp, representing 20.8 Mb of genomic DNA in total or 3.8% of the genome. This database of BAC-end sequences is useful for the assembly of the complete olive flounder genome sequence and is important for identification in functional genomics experiments.