Woodland tobacco (Nicotiana sylvestris, 2n=24) was originally discovered in South America and is largely cultivated nowadays as an ornamental plant. N. sylvestris is a diploid species with an estimated 1C genome size of 2’650 Mb. It belongs to the Solanaceae family and shares significant similarity with tomato, potato, and pepper. It is considered to be the maternal donor which about 200’000 years ago merged through interspecific hybridization with N. tomentosiformis to form N. tabacum, the common tobacco. In plant biology, N. sylvestris serves as a diploid model system for studies of terpenoid production in glandular trichomes or engineering of plastid genomes. In addition, the N. sylvestris genome sequence has the highest identity to the S-genome of N. tabacum and is important for understanding the regulation of gene expression in polyploid species. We collected young leaves of N. sylvestris grown in a greenhouse and constructed genomic and transcriptomic libraries suitable for Next-Generation Sequencing. 44’076’194 paired-end cDNA reads were assembled to build a N. sylvestris leaf transcriptome with ~10’000 full transcripts. An initial assembly was produced from 834’187’700 paired-end, 32’366’838 mate-pair and 64’814’117 single-end genomic reads. This 34x genome assembly was further refined by merging the contigs into supercontigs through mapping onto the tobacco physical map. Here, we report for the first time, the annotation of the draft genome of N. sylvestris. Conclusions resulting from a comparison with genomes of other Solanaceae species will be discussed.