The discovery of DNA regulatory motifs in the sequenced genomes using computational methods remains challenging. Here, we present MotifIndexer--a comprehensive strategy for de novo identification of DNA regulatory motifs at a genome level. Using word-counting methods, we indexed the existence of every 8-mer oligo composed of bases A, C, G, T, r, y, s, w, m, k, n or 12-mer oligo composed of A, C, G, T, n, in the promoters of all predicted genes of Arabidopsis thaliana genome and of selected stress-induced co-expressed genes. From this analysis, we identified number of over-represented motifs. Among these, major critical motifs were identified using a position filter. We used a model based on uniform distribution and the z-scores derived from this model to describe position bias. Interestingly, many motifs showed position bias towards the transcription start site. We extended this model to show biased distribution of motifs in the genomes of both A. thaliana and rice. We also used MotifIndexer to identify conserved motifs in co-expressed gene groups from two Arabidopsis species, A. thaliana and A. lyrata. This new comparative genomics method does not depend on alignments of homologous gene promoter sequences.