We present an integrated toolkit, BoBro2.0, for prediction and analysis of cis-regulatory motifs. This toolkit can (i) reliably identify statistically significant cis-regulatory motifs at a genome scale, (ii) accurately scan for all motif instances of a query motif in specified genomic regions using a novel method for P-value estimation, (iii) provide highly reliable comparisons and clustering of identified motifs, which takes into consideration the weak signals from the flanking regions of the motifs, and (iv) analyze co-occurring motifs in the regulatory regions.