The complete genomic sequence of variant USA6b of Little cherry virus-2 (LChV-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (ORFs). The sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of LChV-2 (variant USA6a). Both LChV-2/USA6a and LChV-2/USA6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. LChV-2/USA6b is more closely related to the partially determined genomic sequence of a Canadian isolate of LChV-2, strain LC5 (92.9% identity). LChV-2/USA6b has an unusual genomic organization compared to other members of the Closteroviridae. The LChV-2/USA6b genome is potentially ambi-sense, with a negative sense ORF0 at the 5' terminus, from which an 18.1 kDa protein of unknown function can be expressed in vitro. The N-terminal region of the LChV-2/USA6 ORF1a translation product does not code for a papain-like protease motif. ORF1 codes for a novel motif, of unknown function, also present in isolates of the Grapevine leafroll associated virus-3, (genus Ampelovirus) as well as viruses of the family Flexiviridae. ORF3 lacks an AUG start codon, but could potentially be expressed via read-through of the ORF2 stop codon. At the 3' end, there is a re-organization of encoded genes compared with other members of the Closteroviridae including separation of the coat protein and coat protein duplicate genes by 4 other genes as found for LChV-2/LC5.