Over-represented k-mers in non-coding genomic regions often lead to identification of potential transcriptional regulatory sites (TRS). This phenomenon has been employed by many algorithms to predict TRS in silico. Yet, the improvement of these algorithms should be based on deeper understanding of the enrichment feature. To obtain a general distributional profile of TRS in different regions of genomes as well as in different genomes, we here performed a systematic analysis on the over-representation of TRS in intergenic regions and gene upstream regions of yeasts and viral genomes, and the distributional pattern of TRS in intergenic and intron regions of the Drosophila genome. We also explored the way to evaluate the accuracy of TRS consensus sequences by measuring their enrichment.