A comparison of uncloned African swine fever virus isolates with their cloned counterparts revealed the presence of genetic variants in three out of seven uncloned field virus populations tested. Five different virus clones were isolated from the uncloned KIR69 virus stock by limit dilution. Structural analysis of the variants showed that they differed by single deletions of 10-16 kilobases in the region located between 6.8 and 27 kilobases from the left DNA terminus. There was no homology between DNA sequences immediately to the left and right of the deletions indicating that the mechanism generating deletion variants was not homologous recombination. The alignment of the restriction maps of the variants with those of other virus isolates indicated that two of the variants lacked the whole multigene family 110. This was confirmed by hybridization of the viral DNA with a degenerate oligonucleotide probe. Each virus variant replicated to high titer and was virulent in domestic pigs. Therefore, the multigene family 110 was not required for viral replication or virulence in domestic pigs. The virus variants were stable upon repeated passage in swine macrophages, indicating that the generation of variants is not a frequent genetic event in vitro.