Abstract Sweet basil, sometimes called the King of Herbs, is well known for its culinary uses, especially in the Italian sauce ‘Pesto’. It is also used in traditional medicine, as a source for essential oils and as an ornamental plant. So far, basil was bred by classical and traditional methods due to lack of a reference genome that will allow optimized application of the most up-to-date sequencing techniques. Here, we report on the first completion of the sweet basil genome of the cultivar ‘Perrie’, a fresh-cut Genovese-type basil, using several next generation sequencing platforms followed by genome assembly with NRGENE’s DeNovoMAGIC assembly tool. We determined that the genome size of sweet basil is 2.13 Gbp and assembled it into 12,212 scaffolds. The high-quality of the assembly is reflected in that more than 90% of the assembly size is composed of only 107 scaffolds. An independent analysis of single copy orthologues genes showed a 93% completeness which reveal also that 74% of them were duplicated, indicating that the sweet basil is a tetraploid organism. A reference genome of sweet basil will enable to develop precise molecular markers for various agricultural important traits such as disease resistance and tolerance to various environmental conditions. We will gain a better understanding of the underlying mechanisms of various metabolic processes such as aroma production and pigment accumulation. Finally, it will save time and money for basil breeders and scientists and ensure higher throughput and robustness in future studies.