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Spinach (Spinacia oleracea L.) is an economically important green leafy vegetable crop. In this study, we performed deep transcriptome sequencing for nine spinach accessions: three from cultivated S. oleracea, three from wild S. turkestanica and three from wild S. tetrandra. A total of approximately 100 million high-quality reads were generated, which were de novo assembled into 72,151 unigenes with a total length of 46.5 Mb. By comparing sequences of these unigenes against different protein databases, nearly 60% of them were annotated and 50% could be assigned with Gene Ontology terms. A total of 387 metabolic pathways were predicted from the assembled spinach unigenes. From the transcriptome sequencing data, we were able to identify a total of ~320,000 high-quality single nucleotide polymorphisms (SNPs). Phylogenetic analyses using SNPs as well as gene expression profiles indicated that S. turkestanica was more closely related to the cultivated S. oleracea than S. tetrandra. A large number of genes involved in responses to biotic and abiotic stresses were found to be differentially expressed between the cultivated and wild spinach. Finally, an interactive online database (http://www.spinachbase.org) was developed to allow the research community to efficiently retrieve, query, mine and analyze our transcriptome dataset.