Next generation sequencing (NGS) technologies have opened fascinating opportunities for the analysis of plants with and without a sequenced genome on a genomic scale. During the last few years, NGS methods have become widely available and cost effective. They can be applied to a wide variety of biological questions, from the sequencing of complete eukaryotic genomes and transcriptomes, to the genome-scale analysis of DNA–protein interactions. In this review, we focus on the use of NGS for plant transcriptomics, including gene discovery, transcript quantification and marker discovery for non-model plants, as well as transcript annotation and quantification, small RNA discovery and antisense transcription analysis for model plants. We discuss the experimental design for analysis of plants with and without a sequenced genome, including considerations on sampling, RNA preparation, sequencing platforms and bioinformatics tools for data analysis. NGS technologies offer exciting new opportunities for the plant sciences, especially for work on plants without a sequenced genome, since large sequence resources can be generated at moderate cost.