Aims: We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P.larvae infections. Methods and Results: For this purpose, we established a protocol for the transformation of the plasmid pAD43-25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re-germination. Conclusions: This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP-tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re-germination. Significance and Impact of the Study: This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.