Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf disease, naturally infects at least three plant species in Florida: sugarcane (Saccharum spp.), the weed Columbus grass (Sorghum almum) and cultivated sorghum (S. bicolor). All three hosts are also colonized by the sugarcane aphid (Melanaphis sacchari), the main vector of SCYLV worldwide. To understand the high incidence of SCYLV observed in sugarcane commercial fields and in germplasm collections, we investigated the transmission efficiency of SCYLV from sugarcane and Columbus grass to sugarcane using the sugarcane aphid and a spider mite (Oligonychus grypus) that also tested positive for SCYLV in Florida. Healthy and SCYLV-infected leaf pieces of sugarcane and Columbus grass carrying viruliferous aphids or spider mites were transferred to virus-free plants of the yellow leaf susceptible sugarcane cultivar CP96-1252. Three- and 6-months post inoculation, the 108 aphid-inoculated plants of Columbus grass and the 90 mite-inoculated plants of sugarcane tested negative for SCYLV by tissue blot immunoassay (TBIA) or reverse transcription polymerase chain reaction (RT-PCR). Similar results were obtained for 162 aphid-inoculated plants of sugarcane, except for two plants that tested positive for SCYLV by TBIA and RT-PCR. In two field experiments planted with SCYLV-free and virus-infected sugarcane (cultivar CP96-1252), only 18-28% of healthy plants became infected during a 24- to 28-month period. SCYLV prevalence in these field experiments did not differ between aphicide treated and untreated plots. Incidence of M. sacchari haplotypes in the Everglades agricultural area also indicated that the predominant haplotype that is currently colonizing sugarcane was not a vector of SCYLV in Florida. Lack of virus transmission by the spider mite suggested that this arthropod only acquired the virus when feeding on infected plants but was unable to transmit SCYLV. The current vector of SCYLV in Florida remains to be identified.