Bacteriophage EP23 that infects Escherichia coli and Shigella sonnei was isolated and characterized. The bacteriophage morphology was similar to members of the family Siphoviridae. The 44,077 bp genome was fully sequenced using 454 pyrosequencing. Comparative genomic and phylogenetic analyses showed that EP23 was most closely related to phage SO-1, which infects Sodalis glossinidius and phage SSL-2009a, which infects engineered E. coli. Genomic comparison indicated that EP23 and SO-1 were very similar with each other in terms of gene order and amino acid similarity, even though their hosts were separated in the level of genus. EP23 and SSL-2009a displayed high amino acid similarity between their genes, but there was evidence of several recombination events in SSL-2009a. The results of the comparative genomic analyses further the understanding of the evolution and relationship between EP23 and its bacteriophage relatives.