British oaks, Quercus robur and Q. petraea, are under threat from Acute Oak Decline (AOD). Severe cases are lethal within five years. Symptoms include necrotic, longitudinal, bleeding cracks in the bark, from which dark fluid emanates. AOD is caused by the interaction of several bacterial species. The lesion microbiome shows a significantly similar composition pattern, where Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana are the predominant pathogens. Species belonging to the Enterobacterales and Pseudomonadaceae are also routinely isolated. Many field samples from affected oak need to be processed on a daily basis, therefore a rapid and cost-effective detection and identification method is needed. A multiplex high resolution melt (HRM) analysis has been developed for the identification of B. goodwinii, G. quercinecans, R. victoriana and Lonsdalea britannica. HRM analysis is a real-time PCR-based technique in which single nucleotide polymorphisms can be identified in amplicons, without DNA sequencing. Our multiplex HRM can identify up to four bacterial pathogens in a single tube in only 40 minutes per HRM run. This diagnostic method will potentially be a useful tool for research institutions working with AOD. To further investigate the bacteria associated with AOD we have identified several novel species of Pseudomonas and described these following a polyphasic approach. Further investigation will establish if the novel Pseudomonas play a role in AOD.