The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales.