Prevalence of Xanthomonas translucens, which causes cereal leaf streak (CLS) in cereal crops and bacterial wilt in forage and turfgrass species, has increased in many regions in recent years. Because the pathogen is seedborne in economically important cereals, it is a concern for international and interstate germplasm exchange and, thus, reliable and robust protocols for its detection in seed are needed. However, historical confusion surrounding the taxonomy within the species has complicated the development of accurate and reliable diagnostic tools for X. translucens. Therefore, we sequenced genomes of 15 X. translucens strains representing six different pathovars and compared them with additional publicly available X. translucens genome sequences to obtain a genome-based phylogeny for robust classification of this species. Our results reveal three main clusters: one consisting of pv. cerealis, one consisting of pvs. undulosa and translucens, and a third consisting of pvs. arrhenatheri, graminis, phlei, and poae. Based on genomic differences, diagnostic loop-mediated isothermal amplification (LAMP) primers were developed that clearly distinguish strains that cause disease on cereals, such as pvs. undulosa, translucens, hordei, and secalis, from strains that cause disease on noncereal hosts, such as pvs. arrhenatheri, cerealis, graminis, phlei, and poae. Additional LAMP assays were developed that selectively amplify strains belonging to pvs. cerealis and poae, distinguishing them from other pathovars. These primers will be instrumental in diagnostics when implementing quarantine regulations to limit further geographic spread of X. translucens pathovars.