This paper describes an evolutionary analysis of IS1 elements based on nucleotide sequence data from six iso-insertion elements of IS1 (iso-IS1s) which are present in chromosomes and plasmids of Escherichia coli and Shigella species as repeated sequences. The sequence comparison, which permitted construction of a phenogram, showed that the iso-IS1s can be divided into three groups. One group consists of four elements with 1% nucleotide sequence divergence. A second and third group each consists of one element, with 10% and 46% divergence, respectively, to the first group. Despite their divergence, amino acid sequences in the two IS1 encoded genes, insA and insB, and the terminal inverted repeat sequences, insL and insR, were found to be highly conserved. The evolutionary distance per site in the six sequences suggests that the IS1 element has diverged to a greater degree than bacterial genes of known nucleotide sequences have. We postulate that the existence of a group of well conserved iso-IS1s and of highly diverged iso-IS1s may be due to the transposition ability of the IS1 element, generating repetitive sequences in both bacterial chromosomes and plasmids which can then independently diverge. We also discuss possible regulatory mechanisms of transposition mediated by IS1 based on this analysis, including the influence of colon usage of insA and insB. The observed colon selection agrees well with those colons used by weakly expressed E. coli proteins.