High-throughput sequencing technology has been widely used and continuously optimized. As one kind of high-throughput sequencing technology, RNA sequencing (RNA-seq)can effectively detect complete transcriptome information, which brings prospects for plant researches. Conifers are a family of Coniferopsida, mainly including Cupressaceae, Taxodiaceae, Pinaceae and Taxaceae trees. Presently, the whole genome sequencing (WGS)of many crops and broadleaf trees have been completed, while that of conifer species are limited due to huge data volume and complicated repeat sequences. Therefore, RNA-seq is one of the most important and practical methods for exploring conifers genes. This review introduced the analysis processes of RNA-seq, and highlighted the applications and prospects of RNA-seq in conifers molecular genetics, which might provide references for future genetic improvement and molecular breeding in conifer species.