Chlorotic fleck, a strawberry disease caused by a graft and aphid transmissible agent, was identified more than 45 years ago in Louisiana. Since its discovery there has been no additional information on the agent that causes the disease. The mode of transmission implies that a virus is the causal agent of chlorotic fleck. We identified four closteroviruses in the single chlorotic fleck infected strawberry clone known to exist in the United States. Sequence analysis indicated that two of the viruses are novel and one of them is closely related to members of the Closterovirus genus, the aphid-transmitted viruses in the family Closteroviridae, a feature that is in accordance with the aphid transmissibility of the chlorotic fleck agent. The genome of the novel Closterovirus, designated as Strawberry chlorotic fleck associated virus exceeds 17 kilobases and encodes 10 open reading frames, including the signature closterovirus genes as well as a gene without obvious homologs in the family. RNA folding predicted a pseudoknot structure near the 3' terminus of the virus that may be involved in template recognition by the viral polymerase. Phylogenetic analysis indicates that Strawberry chlorotic fleck associated virus is most closely related to Citrus tristeza virus among sequenced members of the family. Detection protocols have been developed and the virus was detected in several strawberry plants from production fields.