Fruit species contribute to nutritional and health security by providing micronutrients, antioxidants, and bioactive phytoconstituents, and hence fruit-based products are becoming functional foods presently and for the future. Although conventional breeding methods have yielded improved varieties having fruit quality, aroma, antioxidants, yield, and nutritional traits, the threat of climate change and need for improvement in several other traits such as biotic and abiotic stress tolerance and higher nutritional quality has demanded complementary novel strategies. Biotechnological research in fruit crops has offered immense scope for large-scale multiplication of elite clones, in vitro, mutagenesis, and genetic transformation. Advanced molecular methods, such as genome-wide association studies (GWAS), QTLomics, genomic selection for the development of novel germplasm having functional traits for agronomic and nutritional quality, and enrichment of bioactive constituents through metabolic pathway engineering and development of novel products, are now paving the way for trait-based improvement for developing genetically superior varieties in fruit plant species for enhanced nutritional quality and agronomic performance. In this article, we highlight the applications of in vitro and molecular breeding approaches for use in fruit breeding.