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Grapevines are infected by over 60 different viruses. <i> Grapevine vein clearing virus </i>(GVCV) is associated with a severe vein clearing and vine decline disease that severely affects grape production and berry quality in Midwest vineyards.  Symptoms include translucent vein-clearing, backward rolling, and chlorosis of the leaves as well as short, zig-zagged internodes and a decline of vine vigor. The genome sequence of GVCV-CHA that was isolated from grape cultivar ‘Chardonel’ in a commercial vineyard was published in 2011. It has a circular dsDNA genome of 7,753 bp, and encodes three open reading frames (ORFs) with four short ORFs within the intergenic region. A new isolate of GVCV, referred to as GVCV-VRU, was identified in a wild <i>Vitis rupestris</i> vine in its native site in the southwest region of Missouri. GVCV-VRU associated symptoms are necrotic flecks along major veins in <i>V. rupestris</i>.  Comparative analysis of GVCV-CHA and GVCV-VRU found that GVCV-VRU genome is two base pairs longer than GVCV-CHA genemoe and thyey shared 91.5% identical nucleotides. ORF II is the most variable region with a 9 bp insert only present in GVCV-VRU genome; GVCV-VRU and GVCV-CHA share only 83.3% nucleotide identity. The discovery of a new GVCV isolate in wild grapevine provides important clues on the evolution and epidemics of GVCV.