The bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. In Escherichia coli K-12, the single replication origin oriC is a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequesters oriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entire Escherichia coli K-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in the ter macrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.