Aim: To develop a rapid real-time PCR method for the specific detection and quantification of Bacillus thuringiensis var. israelensis (Bti) spores present in the environment. Methods and Results: Seven soil samples as well as one sediment sample obtained from various regions of Switzerland and characterized by different granulometry, pH values, organic matter and carbonate content were artificially inoculated with known amounts of Bti spores. After DNA extraction, DNA templates were amplified using TaqMan real-time PCR targeting the cry4Aa and cry4Ba plasmid genes encoding two insecticidal toxins (delta-endotoxins), and quantitative standard curves were created for each sample. Physicochemical characteristics of the samples tested did not influence DNA extraction efficiency. Real-time PCR inhibition because of the presence of co-extracted humic substances from the soil was observed only for undiluted DNA extracts from samples with very high organic matter content (68%). The developed real-time PCR system proved to be sensitive, detecting down to 1 x 103 Bti spores per g soil. One-way analysis of variance confirmed the accuracy of the method. Conclusions: Direct extraction of DNA from environmental samples without culturing, followed by a specific real-time PCR allowed for a fast and reliable identification and quantification of Bti spores in soil and sediment. Significance and Impact of the Study: The developed real-time PCR system can be used as a tool for ecological surveys of areas where treatments with Bti are carried out.