The resource consumption and costs for an increased agricultural production can be lowered only due to effective breeding for which biotechnologies and molecular genetics provide powerful tools. Among them, tissue cultures, clonal micropropagation, androgenesis, gynogenesis, and genetic transformation are widely used to obtain diversified forms and homozygous constant lines, and to speed up breeding process (J.M. Dunwell, 2010). Androgenesis and gynogenesis involve individual gametoclonal variability, rare recessive alleles, and unique genetic recombinations into breeding (T. Winkelmann et al., 2006). An interspecific hybridization is a way to develop initial breeding material. Due to it, novel traits (for example, resistance to biotic and abiotic stresses) can be transferred from wild species to cultivated crops, and the range of genetic variability expands (R. Hajjar et al., 2007). An interspecific hybridization incompatibility can be overcome by biotechnological methods, too. Molecular markers are helpful for detecting DNA changes and desired genes’ introgression from one genotype to another, for germplasm fingerprinting, gene mapping, etc. Marker-assisted selection is powerful facility to maintain germplasm collections, to plan crosses, to predict useful gene combination, and to protect varieties and hybrids authenticity. In the review we summarized the results on biotechnology, molecular genetics and their practical use in All-Russian Research Institute of Vegetable Breeding and Seed Production (Moscow Province). In particular, onion (Izumrudnii, Sigma, Zolotye Kupola, Tseparius), salad (Izumrudnii, Tvorets, Aleks, Korall, Malakhit), and physalis (Lakomka, Desertnii) varieties were recently obtained by distant hybridization. Carrot and wild aubergine are involved into interspecial crossings, and pepper plants as sources of resistance to viral infection are selected. Microclonal protocol has been developed for unlimited in vitro propagation of male sterile cabbage. Clonal micropropagation and in vitro cultivation were used to provide embryo viability under interspecial hybridization. A novel protocol developed for in vitro cell cultivation made it possible to create double haploids in carrot and pepper. Due to optimized conditions, double haploids in rape, Chinese cabbage, broccoli and white cabbage were selected. Double haploids of carrots, onion, cucumber, pumpkin, sugar beet, etc., were derived from in vitro cultivated seed-bud without pollination. In this, the ISSR, IRAP, AFLP и SSR markers are involved to assess genome variability and genotyping in vegetable crops.