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To determine the fate of viable Bacillus thuringiensis var. israelensis (Bti) spores dispersed in the environment, using a universally applicable molecular detection methodology.Soil samples were spread on growth medium, after a temperature selection of the spores. A PCR amplification of the cry4Aa and cry4Ba insecticidal genes was applied on the colonies. Ribotyping was performed subsequently. This combined molecular method proved to be very specific for Bti, which was easily differentiated from the other B. thuringiensis serovars. A site regularly treated with Vectobac-G® was chosen within the ‘Bolle di Magadino’ natural reserve, and monitored throughout 1 year for the detection of Bti spores. The results showed that the numbers were relatively high after insecticidal applications (1.4 × 105 CFU g−1), and decreased approx. 10-fold after 220 days. A successive treatment induced a new increase.The results show that yearly repeated use of Vectobac-G® does not seem to have a major ecological impact on the ‘Bolle di Magadino’ natural reserve. Bti spores followed a trend leading to their eventual disappearance from the ecosystem, despite the seasonal application of this biological insecticide for more than a decade.The molecular identification of Bti cells through the PCR analysis of the delta-endotoxins genes coupled to ribotyping, is an innovative method, that has enabled the identification of this organism into wetland environments.