Pathogenicity islands and genomic islands (GI) are key elements in the evolution of bacterial virulence and environmental adaptation. In Salmonella enterica, Salmonella pathogenicity islands (SPI) confer important virulence traits; however, many of these loci have not been characterized in molecular detail. In this chapter, procedures for the identification and molecular characterization of SPI and GI are described. Based on genome sequence data, bioinformatics approaches allow the identification of putative SPI and GI. The role of these loci can be analyzed after the generation of deletion mutant strains using the Red recombination approach. For further analyses, cosmid libraries of S. enterica genomic DNA are screened for clones harboring entire SPI or GI. Such cosmid clones are then used for complementation of SPI or GI deletions as well as for the transfer of these loci to other bacterial species and subsequent functional assays. This set of methods allows the rapid and efficient analyses of the functions of SPI and GI.