AIMS: We assessed the veracity of intergenic spacer region 1 (ITS1) ribotyping for the rapid, inexpensive and accurate identification of Brenneria goodwinii and Gibbsiella quercinecans that are associated with acute oak decline (AOD) in the UK. METHODS AND RESULTS: Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) were applied for the typing of ITS1 PCR amplicons from strains of B. goodwinii, G. quercinecans and related species (n = 34). The number and length of ITS1 amplicons varied significantly between strains. ITS1 profiles generated via PAGE were used to differentiate species using a neighbour-joining phylogram. The ITS1 phylogram was compared against DNA gyrase B (gyrB) gene sequences from the same strains, demonstrating that ITS1 ribotyping is as effective as gyrB at resolving G. quercinecans and B. goodwinii to the species level. CONCLUSIONS: The ITS1 gene has been successfully employed as a novel marker to resolve newly described AOD-associated Enterobacteriaceae, B. goodwinii and G. quercinecans, to species level. SIGNIFICANCE AND IMPACT OF THE STUDY: ITS1 ribotyping of B. goodwinii and G. quercinecans provides equivalent sensitivity to the current standard method for strain identification (sequence analysis of the gyrB gene), but with reduced processing time and cost. Furthermore, the ITS1 gene is widely applicable as a rapid and inexpensive typing system for Enterobacteriaceae.