Objective: Nicotiana glauca (tree tobacco) is a naturally transgenic plant, containing sequences acquired from Agrobacterium rhizogenes by horizontal gene transfer. Besides, N. glauca contains a wide profile of alkaloids of medical interest. Data description: We report a high-depth sequencing and de novo assembly of N. glauca full genome and analysis of genome elements with bacterial origin. The draft genome assembly is 3.2 Gb, with N50 size of 31.1 kbp. Comparative analysis confirmed the presence of single, previously described gT insertion. No evidence was acquired to support idea of multiple T-DNA insertions in the N. glauca genome. Our data is the first comprehensive de novo assembly of tree tobacco and provide valuable information for researches in pharmacological and in phylogenetic fields.