Insertion sequences (IS) are small, mobile genetic elements (MGE) that can elicit a wide range of effects on bacterial genomes. The current literature surrounding IS focuses mainly on their effect in single genomes, with only speculative knowledge regarding the effect of IS from a phylogenetic perspective. Here, we aimed to further investigate the role of IS from an evolutionary standpoint using a large dataset of 95 strains within the globally disseminated multidrug resistant uropathogenic Escherichia coli ST131 lineage. IS annotated in the completely sequenced E. coli ST131 strain EC958 were used to broadly determine IS conservation throughout the ST131 lineage using the visualization tool SeqFindr, which indicated that IS were highly conserved. We then developed a novel method to identify IS1 locations, IS_Locator, which uses paired-end read information to resolve contig gaps caused by collapsed repeats during genome assembly. Our method identified 82 novel IS1 locations, which were found to be preferentially located within chromosomal regions proximal or in association with MGE (p < 0.0001). We also experimentally determined the phenotypic effect of several novel IS1 locations, however, all were determined to be isolated events within specific ST131 strains and did not contribute to the lineage as a whole. In summary, while IS have previously been ignored in phylogenetic analyses due to difficulties assembling them into complete genomes, we hypothesize that their role in the evolution of bacterial lineages may be more profound than previously thought. Further investigation into the phylogenetic relevance of IS will enable a more complete understanding of their impact on the ST131 lineage.