Sugarcane mosaic virus (SCMV) was detected by reverse transcription polymerase chain reaction in eight different species of the Poaceae family in the Everglades Agricultural Area (EAA) of south Florida: broadleaf signalgrass (Urochloa platyphylla), Columbus grass (Sorghum almum), goosegrass (Eleusine indica), maize (Zea mays), sorghum (Sorghum bicolor), St. Augustine grass (Stenotaphrum secundatum), southern crabgrass (Digitaria ciliaris), and sugarcane (Saccharum interspecific hybrids). Based on their coat protein (CP) gene sequence, 62 isolates of SCMV from Florida and 29 worldwide isolates representing the known genetic diversity of this virus were distributed into eight major phylogenetic groups. SCMV isolates infecting Columbus grass, maize, and sorghum in Florida formed a unique group, whereas virus isolates infecting sugarcane in the United States (Florida and Louisiana) clustered with isolates from other countries. Based on the entire genome coding region, SCMV isolates infecting sugarcane in Florida were closest to virus isolates infecting sorghum species or St. Augustine grass. Virus isolates from Columbus grass, St. Augustine grass, and sugarcane showed different virulence patterns after mechanical inoculation of Columbus grass, St. Augustine grass, and sugarcane plants, thus proving that these isolates were different pathogenic strains. Sugarcane was symptomless and tested negative for SCMV by tissue blot immunoassay after inoculation with crude sap from SCMV-infected Columbus grass, indicating that Columbus grass was not a reservoir for SCMV infecting sugarcane in the EAA. Close CP sequence identity between isolates of SCMV from Columbus grass, maize, and sorghum suggested that the same virus strain was naturally spreading between these three plants in south Florida.