Biotechnological tools such as clonal propagation, haploid production, protoplast fusion, marker-assisted selection, and quantitative trait loci mapping are the age-old approaches in commercial forestry. Biotechnology has shown immense guarantee for the subsequent generation of plant breeders to alleviate the rising demand for food, fiber, and wood. The probable social and environmental impacts of the release of transgenic trees become a progressively more debatable issue and call more considerations. The present review is an attempt to summarize how advances in molecular biology i.e., comparative genomics and genome assembly approaches identify numerous polymorphic molecular markers like microsatellite, simple sequence repeats, single-nucleotide polymorphism (SNP), random amplified polymorphic DNA etc. and revolutionize the science of forest tree biotechnology during the last decade. The advent of next-generation sequencing technology identified many microsatellite loci in forest trees. Microsatellite markers play an important role in population genetics, conservation ecology, and phylogenetic analysis. SNP markers provide novel opportunities for the discovery of regulatory elements in the genome and eventually accelerate forest tree breeding.