Insertion sequences (ISs) are mobile genetic elements widely distributed among bacteria. Their impact on the bacterial genome is multifold, including transfer of genetic information, shuttle of adaptive traits and influence on the genomic content. As a result, ISs play an important role in the organization, plasticity and evolution of bacterial genomes. In this study, four new IS elements: ISMbov7; ISMbov4 and ISMbov5; and ISMbov6, related, respectively, to the IS3, IS4 and IS30 gene families, were identified and characterized with respect to inverted repeat (IR) and directly repeated (DR) sequences, putative target specificity and motifs related to transposase function. For instance, IS30-related ISMbov6 isoform elements were shown to (1) contain an alpha-helix-turn-alpha-helix homeodomain (HTH), (2) generate long DR and (3) possess target specificity for a palindromic sequence derived from putative rho-independent transcription terminators. Members of the IS3 family, which had not been documented previously in Mycoplasma bovis, contain HTH, leucine zipper and AT-hook motifs, which may be involved in DNA binding. In addition, the availability of the M. bovis PG45 genome sequence allowed us to elucidate the genomic organization of 54 intact or truncated IS elements and their possible effect on the expression of adjacent genes.