Transcription factors are key regulatory elements that control gene expression. Recognition of transcription factor binding site (TFBS) motifs in the upstream region of coexpressed genes is therefore critical towards a true understanding of the regulations of gene expression. The task of discovering eukaryotic TFBSs remains a challenging problem. Here, we demonstrate that evolutionary computation can be used to search for TFBSs in upstream regions of genes known to be coexpressed. Evolutionary computation was used to search for TFBSs of genes regulated by octamer-binding factor and nuclear factor kappa B. The discovered binding sites included experimentally determined known binding motifs as well as lists of putative, previously unknown TFBSs. We believe that this method to search nucleotide sequence information efficiently for similar motifs will be useful for discovering TFBSs that affect gene regulation.