Bacterial leaf streak (BLS) has been a major disease on wheat and barley in the Northern Great Plains. It is caused by Xanthomonas translucens pv. undulosa (Xtu) on wheat and X. translucens pv. translucens (Xtt) on barley. Many questions remain unclear on pathogen biology and BLS epidemiology. Based on previous study, I identified an Xtu/Xtt specific region and established a qPCR quantification method for the bacterial pathogens. The method was shown to be effective to detect and quantify the bacterial pathogen in seeds and leaves. In addition, molecular markers were developed to differentiate Xtu and Xtt. Those markers were successfully used to characterize a collection of X. translucens strains into Xtu or Xtt. The results were also confirmed by pathogenicity tests on wheat and barley. The efficient Xtt/Xtu quantification and differentiation methods will be powerful tools to study disease epidemiology and host pathogen interaction for the two bacterial pathogens.