The aim of this study is to improve the breeding efficiency of new seedless grape germplasm with disease resistance and high quality, and lay a foundation for further research on Chinese wild grape germplasms. The seedless European grape varieties were used as the female parent, while the Chinese wild grape germplasms ‘Tangwei’, ‘Xuefeng’, Vitis amurensis Rupr. Cv ‘Shuangyou’, and ‘Beichun’ (Vitis vinifera L. × Vitis amurensis Rupr.) were used as the male parent. The sampling time and culture medium were optimized using embryo rescue techniques, and the progeny were screened for seedless and disease-resistant traits using molecular markers. The results showed that: (1) a total of 13,341 hybrid berries were obtained from 15 hybrid combinations, among which 8,589 young ovules were inoculated, and 1,338 young embryos were developed, with an average embryo development rate of 15.59%; 234 normal seedlings were established, with an average seedling establishment rate of 2.73%; the combination of ‘Ruby Seedless’× ‘Tangwei’ had the best embryo rescue effect with 40.17% embryo development rate, 7.76% seedling establishment rate and 178 obtained plants; (2) at 42 d after pollination, the development rate and germination rate for hybrid embryos of ‘Flame Seedless’ × ‘Beichun’ reached the peak, which were 11.37% and 3.52%, respectively; furthermore, at 38 d after pollination, the development rate and germination rate for hybrid embryos of ‘Jingkejing’ × ‘Beichun’ reached the highest level, which were 4.3% and 1.08%, respectively; (3) the hybrid ovules of ‘Ruby Seedless’ × ‘Tangwei’ were cultured on MM3 medium with exogenous addition of 3 mmol/L putrescine, the embryo development rate and seedling establishment rate achieved the peak level, which were 55.83% and 20%, respectively; (4) the ovules of the four naturally pollinated varieties ‘Ruby Seedless’, ‘Beauty Seedless’, ‘Kunxiang Seedless’ and ‘Heshi Seedless’ were cultured in different embryos development media, and the E20A medium was more conducive to improve the development rate of young embryos; (5) by utilizing the seedless grape markers SCF27-2000 and GSLP1-569, downy mildew-resistant markers S294-369 and S382-615, and powdery mildew-resistant marker OPY13-661 for screening, 16 hybrid offspring simultaneously carried the markers of seedless, downy mildew resistance and powdery mildew resistance. This experiment could provide new materials for resistance breeding of seedless grape varieties.