2-Methylketones are accumulated in some plants as a byproduct of the fatty acid biosynthesis that takes place in plastids. Methylketone synthase 2 (MKS2) is a thioesterase that catalyzes the penultimate reaction in the methylketone biosynthetic pathway. It converts 3-ketoacyl-ACPs into 3-ketoacids which are precursors for the synthesis of 2-methylketones. Previous studies have shown that species in the Solanaceae family might have different number of MKS2 genes. Besides, each MKS2 could hydrolyze a specific group of endogenous fatty acyl-acyl carrier protein substrates that varies in chain length (C6-C18), degree of saturation and oxidation state. In this study, we had identified two homologous genes of ShMKS2, designated as NtMKS2-1 and NtMKS2-2, located at two contigs AWOJ-SS748 and AWOJ-SS6425 in the Nicotiana tabacum genome database. Both genes comprise of five exons and four introns. The full-length cDNA sequences encoding NtMKS2-1 and NtMKS2-2 have been successfully isolated from young leaf tissues of tobacco N. tabacum, sequenced and aligned with the corresponding sequences predicted by in silico analysis. Both of the deduced amino acid sequences encode proteins of 24 kDa that share more than 60% identity to ShMKS2 and contain a conserved aspartate residue essential to the catalytic core of the hotdog-fold thioesterases. This study provided additional data to gain insights into the evolution of the MKS2 genes in species from the Solanaceae family.