RNA-seq has become a state-of-the-art technique for transcriptomic studies. Advances in both RNA-seq techniques and the corresponding analysis tools and pipelines have unprecedently shaped our understanding in almost every aspects of plant sciences. Notably, the integration of huge amount of RNA-seq with other omic data sets in the model plants and major crop species have facilitated plant regulomics, while the RNA-seq analysis has still been primarily used for differential expression analysis in many less-studied plant species. To unleash the analytical power of RNA-seq in plant species, especially less-studied species and biomass crops, we summarize recent achievements of RNA-seq analysis in the major plant species and representative tools in the four types of application: (1) transcriptome assembly, (2) construction of expression atlas, (3) network analysis, and (4) structural alteration. We emphasize the importance of expression atlas, coexpression networks and predictions of gene regulatory relationships in moving plant transcriptomes toward regulomics, an omic view of genome-wide transcription regulation. We highlight what can be achieved in plant research with RNA-seq by introducing a list of representative RNA-seq analysis tools and resources that are developed for certain minor species or suitable for the analysis without species limitation. In summary, we provide an updated digest on RNA-seq tools, resources and the diverse applications for plant research, and our perspective on the power and challenges of short-read RNA-seq analysis from a regulomic point view. A full utilization of these fruitful RNA-seq resources will promote plant omic research to a higher level, especially in those less studied species.