Seedlessness in grape (Vitis vinifera) is an important commercial trait for both the fresh and drying markets.  However, despite numerous studies, the mechanisms and key genes regulating grape seedlessness are mostly unknown.  In this study, we sequenced the genomes of the V. vinifera seeded cultivar ‘Red Globe’, the seedless cultivar ‘Centennial Seedless’, and the derived hybrids.  Nonsynonymous single nucleotide polymorphisms (SNPs) were identified by genome sequencing and analyzed using published transcriptome data.  Nonsynonymous SNPs occurred in genes related to seed development, which were identified as protein kinases, transcription factors, and cytochrome P450s and showed differential expression during ovule development in both seeded and seedless grapes.  These nonsynonymous SNP-associated genes were mainly involved in biological processes such as hormone balance, seed coat and endosperm development, reproductive organ development, oxidation and reduction, senescence and cell death.  A potential quantitative trait locus (QTL) region associated with seed size was characterized based on the SNP-index, and expression analysis of candidate genes in the QTL region during ovule development in multiple seeded and seedless grape cultivars were conducted.  Three SNPs were further subjected to SNaPshot analysis and one SNP in G8 showed 67.5% efficiency in the grape progeny validation.  Overall, the data obtained in this study shed light on the differences in seed development between seeded and seedless progeny at the genomic level, which provides valuable resources for future functional studies and grape breeding.