MADS-box transcription factors are involved in many aspects of plant growth and development, such as floral organ determination, fruit ripening, and embryonic development. Yet not much is known about grape (Vitis vinifera) MADS-box genes in a relatively comprehensive genomic and functional way during ovule development. Accordingly, we identified 54 grape MADS-box genes, aiming to enhance our understanding of grape MADS-box genes from both evolutionary and functional perspectives. Synteny analysis indicated that both segmental and tandem duplication events contributed to the expansion of the grape MADS-box family. Furthermore, synteny analysis between the grape and Arabidopsis genomes suggested that several grape MADS-box genes arose before divergence of the two species. Phylogenetic analysis and comparisons of exon-intron structures provided further insight into the evolutionary relationships between the genes, as well as their putative functions. Based on phylogenetic tree analysis, grape MADS-box genes were divided into type I and type II subgroups. Tissue-specific expression analysis suggested roles in both vegetative and reproductive tissue development. Expression analysis of the MADS-box genes following gibberellic acid (GA3) treatment revealed their response to GA3 treatment and that seedlessness caused by GA3 treatment underwent a different mechanism from that of normal ovule abortion. Expression profiling of MADS-box genes from six cultivars suggests their function in ovule development and may represent potential ovule identity genes involved in parthenocarpy. The results presented provide a few candidate genes involved in ovule development for future study, which may be useful in seedlessness-related molecular breeding programs.