Bacterial diseases affecting wheat production worldwide have been an issue of growing concern in the last decade. Nevertheless, the main bacterial diseases of wheat in Uruguay remain largely uninvestigated. To identify bacterial pathogens associated with diseased wheat fields, 61 fields were surveyed from 2017 to 2019 in western Uruguay, yielding a collection of strains identified as Xanthomonas spp. by 16S rDNA sequencing. These strains were further characterized via multilocus sequence analysis (MLSA) and typing (MLST), as well as in planta pathogenicity assays. MLSA grouped 44 strains with reference strains for Xanthomonas translucens pv. undulosa, the pathovar predominantly associated with bacterial leaf streak of wheat (BLS). To evaluate the genetic diversity among strains, MLST was applied, revealing a low diversity among Uruguayan strains identified as X. translucens pv. undulosa. In addition, 17 strains in the collection were assigned to a separate clade distant from the X. translucens species, grouping together with previously unreported Xanthomonas strains isolated from wheat in Minnesota, USA. In planta pathogenicity assays were performed on BLS susceptible wheat seedlings. The 44 X. translucens pv. undulosa strains caused greasy, dark brown necrosis symptoms typical of BLS, while the 17 non-translucens Xanthomonas sp. strains caused distinctly different dry tan necrosis symptoms. These results reveal that the main bacterial pathogen affecting wheat crops in Uruguay is X. translucens pv. undulosa, the causal agent of BLS; and that other pathogenic Xanthomonas species are associated with wheat crops in South and North America.