All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non-essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a dominant factor controlling the genetic footprint of transposons in living cells.