Comparative sequence analysis methods, such as phylogenetic footprinting, represent one of the most effective ways to decode regulatory sequence functions based upon DNA sequence information alone. The laborious task of assembling orthologous sequences to perform these comparisons is a hurdle to these analyses, which is further aggravated by the relative paucity of tools for visualization of sequence comparisons in large genic regions. Here, we describe a second-generation implementation of the GenePalette DNA sequence analysis software to facilitate comparative studies of gene function and regulation. We have developed an automated module called OrthologGrabber (OG) that performs BLAT searches against the UC Santa Cruz genome database to identify and retrieve segments homologous to a region of interest. Upon acquisition, sequences are compared to identify high-confidence anchor-points, which are graphically displayed. The visualization of anchor-points alongside other DNA features, such as transcription factor binding sites, allows users to precisely examine whether a binding site of interest is conserved, even if the surrounding region exhibits poor sequence identity. This approach also aids in identifying orthologous segments of regulatory DNA, facilitating studies of regulatory sequence evolution. As with previous versions of the software, GenePalette 2.1 takes the form of a platform-independent, single-windowed interface that is simple to use.