Maize Streak Mastrevirus (MSV) isolates differ in the severity of symptoms they incite on maize. Virulent forms of the virus incite severe stunting and chlorosis on infected plants and drastically reduce yield. Of major importance to breeding programs is the knowledge of whether current resistance is durable when challenged with the most virulent MSV isolates. However, the relative virulences of MSV isolates across the geographic range of MSV is unclear. A reliable method for identifying MSV virulent types would, therefore, be of considerable value. We focused on developing a means of identifying virulent MSV isolates based upon genetic variation. The genomes of four isolates were cloned and sequenced. Dimeric cloned DNA of each isolate was infective when inoculated to susceptible maize by vascular puncture inoculation. Clone symptom severity was classified as mild (MSV-KL), moderately mild (MSV-Z), moderate (MSV-KH, or severe (MSV-Km). Virulence determinants were mapped using chimeric clones constructed from parental clones pMSV-KL (mild) and pMSV-Km (severe) employing restriction endonuclease fragments. Complementary exchange of restriction fragments between the genomes of pMSV-Km (severe) and pMSV-KL (mild) demonstrated that determinants of virulence were present in several regions of the genome, including those encoding the coat protein, replication associated and replication initiator proteins and the non-coding long and short intergenic regions. Analyses of these sequences, along with MSV sequences from the GenBank for isolates with reported symptom phenotypes, were conducted for motifs associated with virulence. The analysis involved various on-line tools and databases. Phylogenetic analyses of the amino acid sequences of the replication-associated (Rep and RepA), movement and coat proteins failed to reveal distinct African regional strains or symptom phenotype motifs. It was concluded that MSV isolates from Africa exist as a common, multi-allelic genome population without a simple genetics of virulence.