Grapevine yellow speckle viroid-2 (GYSVd-2) is a viroid found only in grapevines in China and Australia. Here, we report the molecular characterization of GYSVd-2 isolated from three grapevine varieties in China. A total of 90 cDNA clones were sequenced including 30 cDNA clones obtained from each of the Black Olympia, Zaoyu, and Thomson Seedless isolates. Sequencing analysis identified 20, 18, and 12 different sequence variants from the 3 isolates, respectively. Furthermore, each of the isolates included one predominant sequence variant. Compared to the Australian variant of GYSVd-2 (Accession number: NC_003612), the Black Olympia variant was identical and the Zaoyu variant contained one substitution. In contrast, the Thomson Seedless isolate significantly varied from the Australian variant with three substitutions, two insertions, and four deletions. In silico structure analysis predicted that the variations were clustered in the terminal left, the pathogenicity, and the variable region of the predicted secondary structure of GYSVd-2.