Polynucleotide sequence relatedness in strains of Shigella species was assessed by determining the extent of reassociation in heterologous deoxyribonucleic acid preparations. Thermal elution chromatography on hydroxyapatite was used to separate reassociated nucleotide sequences from nonreassociated sequences and to determine the degree of unpaired bases within related nucleotide sequences. Almost all Shigella strains share 80% or more of their nucleotide sequences. Less than 3% of unpaired bases are present in these related sequences. The same extent of relatedness is present between Shigella and Escherichia coli strains. Strains of S. boydii C13 are highly interrelated. These strains average only about 65% relatedness to other Escherichieae. We were unable to detect preferentially high relatedness between those Shigella and E. coli strains that contain identical or related O antigens.