The Solanaceae family includes many species of plants with high nutritional and medicinal value. Plants in this family have evolved towards diversifying specialized (secondary) metabolism to adapt to adverse conditions, and a few of them have been used as model plants in the study of plant defense. 2-Methylketones are insecticidal compounds that accumulate in certain plants, particularly in wild tomato Solanum habrochaites f. glabratum – a Solanaceae member. 2-Methylketones are the decarboxylated products of 3-ketoacids generated through the hydrolysis of 3-ketoacyl-ACPs by methylketone synthase 2 (MKS2). In this study, we isolated one of the four MKS2 homolog genes from the cultivated potato Solanum tuberosum and designated it as StMKS2-3. A combined in silico approach including sequence alignment, phylogenetic assessment, 3D structural modeling and RNA-sequencing data analysis was performed to exploit the functional properties of this gene. The encoded protein possesses the conserved Aspartate and functional domain characteristic of single Hotdog-fold thioesterases, and it shares high similarity in sequence and structure with SlMKS2a of the cultivated tomato (S. lycopersicum). This suggests that, like SlMKS2a, StMKS2-3 could act as a 3-ketoacyl-ACP thioesterase. According to RNA-seq data, StMKS2-3 exhibited higher expression than the other three StMKS2 genes in most tissues across different potato S. tuberosum cultivars. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that StMKS2-3 was expressed in multiple potato tissues, both aerial and root parts, but the highest expression was in tubers and sprouts. Furthermore, this gene appeared to be transcriptionally induced in response to salinity, drought, and Phytophthora infestans infection, supporting a possible role for StMKS2-3 in the response of potato S. tuberosum to such stress.