One of the major objectives of table grape breeding is to produce seedless grapes of Vitis vinifera L. Of high importance among these objectives is the development of new cold-resistant seedless grapes for the colder regions of the world. Embryo rescue is an effective tool in breeding seedless grapes. Here, we report on nine cross-combinations between seedless cultivars and cold-resistant seedless cultivars or seeded hybrids. We carried out embryo rescue and molecular marker-assisted selection to create new cold-resistant seedless grape germplasm. We also examined the effects of different parents and the use of exogenous hormones on the success of embryo rescue as well as the optimal sampling time for a new seedless cultivar Qinxiu. The results show that a total of 473 new grape genotypes were obtained by embryo rescue using double-phase MM3 (modified ER) as the medium for embryo development and solid phase WPM as the medium for embryo germination and plantlet formation. We found the seedless cultivars Perlette, Qinhong No.2, Ruby Seedless and Qinhong No.10 were the most suitable female parents for embryo rescue, and the cold-resistant seedless cultivar Jupiter (a V. vinifera × V. labrusca hybrid) as the male parent. This was better than the seeded hybrid 0-1-5 (V. vinifera × V. amurensis). The best embryo development medium was MM3 with 500 mg/L CH, 1 mmol/L serine, 0.5 mg/L GA3, 1.0 mg/L IAA and 0.5 mg/L 6-BA. The best embryo germination and plantlet formation medium was WPM with 0.2 mg/L 6-BA and 0.1 mg/L IAA. The best sampling time for Qinxiu for embryo rescue was 42 days after flowering. We obtained a total of 440 hybrids by embryo rescue using the seedless molecular marker SCF27-2000. Hybridization between grape seedless cultivars and cold-resistant cultivars and embryo rescue were performed. We obtained 473 new germplasms and performed marker assisted-selection using SCF27-2000. The embryo rescue system was optimized.