Streptococcus thermophilus bacteria are used as a starter in the fermentation of yogurts and many cheeses. To construct mutants of S. thermophilus CNRZ368, the use of the plasmid pGh9:ISS1 was considered. This plasmid is known to be a good tool for insertional mutagenesis in gram-positive bacteria, owing to its ability to integrate in the genome by a mechanism of replicative transposition. However, the presence of three endogenous ISS1 copies in the ge-nome of S. thermophilus CNRZ368 and the possible occurrence of homologous recombination could reduce the efficiency of pGh9:ISS1 as a tool for generating mutants. To address this question, the ability of pGh9:ISS1 to transpose randomly in the genome of strain CNRZ368 was investigated. The results of our experiments indicated that: (i) the frequency of transposition of ISS1 was high, approximately 2 × 10 –2 , in S. thermophilus CNRZ368; (ii) the integration of multiple tandem copies of the plasmid was frequent; (iii) homologous recombination events between ISS1 were not predominant; and (iv) plasmid pGh9:ISS1 transposed randomly around the S. thermophilus CNRZ368 chromosome. In addition, we describe the strategy used to localize the pGh9:ISS1 insertion locus on the physical map of strain CNRZ368 and the method used to clone the regions flanking this insertion site, especially when multiple copies of the plasmid were integrated in tandem.