The genus Vitis (the grapevine) is a group of highly diverse, diploid woody perennial vines consisting of approximately 60 species from across the northern hemisphere. It is the world’s most valuable horticultural crop with ~8 million hectares planted, most of which is processed into wine. To gain insights into the use of wild Vitis species during the past century of interspecific grape breeding and to provide a foundation for marker-assisted breeding programmes, we present a principal components analysis (PCA) based ancestry estimation method to calculate admixture proportions of hybrid grapes in the United States Department of Agriculture grape germplasm collection using genome-wide polymorphism data. We find that grape breeders have backcrossed to both the domesticated V. vinifera and wild Vitis species and that reasonably accurate genome-wide ancestry estimation can be performed on interspecific Vitis hybrids using a panel of fewer than 50 ancestry informative markers (AIMs). We compare measures of ancestry informativeness used in selecting SNP panels for two-way admixture estimation, and verify the accuracy of our method on simulated populations of admixed offspring. Our method of ancestry deconvolution provides a first step towards selection at the seed or seedling stage for desirable admixture profiles, which will facilitate marker-assisted breeding that aims to introgress traits from wild Vitis species while retaining the desirable characteristics of elite V. vinifera cultivars.