Trehalose 6-phosphate (Tre6P) is a signal metabolite that links carbon metabolism with plant development. Our current understanding of Tre6P metabolism and signalling is predominantly based on studies in Arabidopsis thaliana. Some features could be adapted to the specific physiology, anatomy, and life cycle of this C3 eudicot model species, and thus might not be representative of other angiosperms. To better understand Tre6P metabolism in monocot C4 species we used Setaria viridis, which has been widely adopted as a model for the major C4 NADP-malic enzyme subtype crop species, such as maize (Zea mays), sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum). In this work, we analysed the levels of transcripts encoding Tre6P-related enzymes in different tissues and cell types from S. viridis. The TREHALOSE-6-PHOSPHATE SYNTHASE1 transcript, its encoded protein (TPS1, the enzyme responsible for Tre6P synthesis) and Tre6P were mainly located in bundle sheath cells of S. viridis. Our results show that Tre6P is predominately synthesized and located in bundle sheath and associated cells.