Nucleotide sequence comparison of the internal region of the ati gene of members of the Orthopoxvirus genera revealed that this gene is variable among different species, although within members of the same species it is considered to be well conserved. Previous studies indicated that there is genetic variability in the ati gene among some Brazilian Vaccinia virus strains. To further investigate this variability, we performed molecular analysis of the internal region of the ati gene of eight Brazilian Vaccinia virus strains. While the internal region of this gene in one strain was similar to the Western Reserve strain, four strains presented two blocks of deletions in the analyzed region, and the ati gene was almost entirely deleted from three other strains. These findings demonstrate that there is genetic polymorphism within the ati gene among different Brazilian Vaccinia virus strains.