Slender wild oat (Avena barbata) is an annual grass dominant in many grassland ecosystems in Mediterranean climate. This species has been the subject of ecological studies aimed at understanding the effect of global climate change on grassland ecosystems and the genetic basis for adaptation under varying environmental conditions. We present the sequencing and analysis of cDNA libraries constructed from leaf and root samples collected from A. barbata grown on natural soil and under varying rainfall patterns. More than 1 million expressed sequence tags (ESTs) were generated using both GS 454-FLX pyrosequencing and Sanger sequencing, and these tags were assembled into consensus sequences. We identified numerous candidate polymorphic markers in the data set, providing possibilities for linking the genomic and the existing genetic information for A. barbata. Using the digital Northern method, we showed that genes involved in photosynthesis were down-regulated under high rainfall while stress-related genes were up-regulated. We also identified a number of genes unique to the root library with unknown function. Real-time reverse transcription-PCR was used to confirm the root specificity of some of these transcripts such as two genes encoding O-methyl transferase. Also we showed differential expression of five root-specific genes under three water levels and two developmental stages. Through a combination of Sanger and 454-based sequencing technologies, we were able to generate a large set of transcribed sequences for A. barbata. This data set provides a platform for further studies of this important wild grass species.