Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.