A collection of 38 Lactobacillus helveticus strains, isolated from a number of different artisan Italian cheeses, and 4 reference strains were studied with respect to the presence of insertion sequences and their distribution and abundance. The mobile genetic element ISLh1, that contains one open reading frame coding for a putative transposase of the IS982 family, was used for DNA fingerprinting, together with IS1201 and ISL2, previously isolated from L. helveticus. The number of insertion sequences per strain and the size of DNA restriction fragments containing them, was variable and allowed the discrimination at the strain-level. The genomic distribution of the three unrelated insertion sequences showed significant correlations and allowed the differentiation of the strains also with regard to the specific ecological niche of origin of the isolates. Consequently, insertion sequences comparison may be useful in determining the history of a group of strains known to be related because of identity and offers a further parameter for evaluating the population polymorphism in L. helveticus.