Bacterial leaf streak caused by different pathovars of Xanthomonas translucens is the most important seedborne bacterial disease of small grain cereals. However, variations in the virulence-associated genomic areas of the pathogen remain uninvestigated. In this study, the diversity of transcription activator-like effectors (TALE) was investigated using the Southern blotting of BamHI-digested genomic DNAs in the Iranian strains of X. translucens. All 65 X. translucens strains were assigned into 13 genotypes, where 57 X. translucens pv. undulosa strains were placed in genotypes 1 to 8, and seven X. translucens pv. translucens strains were placed in genotypes 9 to 12. Interestingly, we did not find any TALE genes in the strain XtKm7 (genotype 13), which showed to be pathogenic only on barley. Virulence and aggressiveness of these strains in greenhouse conditions were in agreement with the TALE-based clustering of the strains in the pathovar level, though variations were observed in the aggressiveness of X. translucens pv. undulosa strains. In general, strains containing higher numbers of putative TALE genes were more virulent on wheat and barley than strains containing fewer. This is the first TALE-based genetic diversity analysis on X. translucens strains and provides novel insights into the virulence repertories and genomic characteristics of the pathogen. Further investigations using TALE mutagenesis and complementation analysis are warranted to precisely elucidate the role of each detected X. translucens TALE in bacterial virulence and aggressiveness either on wheat or barley.