Previously, we described a highly conserved nonessential African swine fever virus (ASFV) right variable region gene, NL. Deletion of NL from the European pathogenic isolate E70 resulted in almost complete attenuation of the virus in domestic swine. To study gene function further, NL gene deletion mutants were constructed from two pathogenic African ASFV isolates, Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Pr4). Unexpectedly, both Mal (Mal-deltaNL) and PR4 (Pr4deltaNL) null mutants remained highly virulent when inoculated in swine. Mal-deltaNL exhibited a disease and virulence phenotype indistinguishable from its revertant, Mal-NLR, which caused 100% mortality. Mortality among Pr4deltaNL-infected animals was also high; however, a significant delay in onset of fever and viraemia and in time to death was observed. These data indicate that NL gene function is not required for ASFV virulence and that other yet-to-be identified viral determinants perform significant virulence functions in these African field isolates.