Comparative genomics is a powerful approach for inferring the history and function of genomic sequence. The generation of bacterial artificial chromosome (BAC)-based physical maps is a proven method for the targeted comparative genomic analysis of genes or regions of interest. ‘Universal’ overgo hybridization probes can be used for the efficient construction of BAC-based physical maps of orthologous chromosome segments from multiple species in parallel. ‘Universal’ overgo hybridization probes can therefore facilitate the assembly of deep and diverse collections of experimental and computational comparative genomic resources corresponding to specific segments of the genome. The design of ‘universal’ overgo probes is dependent on the presence of sequences that are highly conserved within a group of species. Such conserved sequences can be readily identified using local-or whole-genome interspecies sequence alignments. Once ‘universal’ overgo hybridization probes are designed, simple and uniform labeling and hybridization conditions can be carried out to exploit the utility of these probes for targeted comparative physical mapping.