ABSTRACT Maize streak virus (MSV) is best known as the causal agent of maize streak disease. However, only a genetically uniform subset of the viruses within this diverse species is actually capable of producing severe symptoms in maize. Whereas these "maize-type" viruses all share greater than 95% sequence identity, MSV strains isolated from grasses may share as little as 79% sequence identity with the maize-type viruses. Here, we present the complete genome sequences and biological characterization of two MSV isolates from wheat that share approximately 89% sequence identity with the maize-type viruses. Clonal populations of these two isolates, named MSV-Tas and MSV-VW, were leafhopper-transmitted to Digitaria sanguinalis and a range of maize, wheat, and barley genotypes. Whereas the two viruses showed some differences in their pathogenicity in maize, they were both equally pathogenic in D. sanguinalis and the various wheat and barley genotypes tested. Phylogenetic analyses involving the genome sequences of MSV-Tas and MSV-VW, a new maize-type virus also fully sequenced in this study (MSV-VM), and all other available African streak virus sequences, indicated that MSV-Tas and MSV-VW are close relatives that together represent a distinct MSV strain. Sequence analyses revealed that MSV-VM has a recombinant genome containing MSV-Tas/VW-like sequences within its movement protein gene.