A novel custom microarray for largemouth bass (LMB) Micropterus salmoides was designed with sequences obtained from a normalized cDNA library using the 454 Life Sciences GS-20 pyrosequencer. This approach yielded in excess of 58 million bases of high-quality sequence. The sequence information was combined with 2616 reads obtained by traditional suppressive subtractive hybridizations to derive a total of 31 391 unique sequences. Annotation and coding sequences were predicted for these transcripts where possible. About 15 950 annotated transcripts were selected as target sequences for the design of the custom LMB oligonucleotide microarray. The microarray was validated by examining the transcriptomic response in male LMB exposed to 17β-oestradiol. Transcriptomic responses were assessed in liver and gonad and indicated gene expression profiles typical of exposure to oestradiol. The results demonstrate the potential to rapidly create the tools necessary to assess large-scale transcriptional responses in non-model species, paving the way for expanded impact of toxicogenomics in ecotoxicology.