Plants trigger endoplasmic reticulum (ER) pathways to survive stresses, but the assistance of ER in plant tolerance still needs to be explored. Thus, we selected sensitive and tolerant contrasting abiotic stress sorghum varieties to test if they present a degree of tolerance to ER stress. Accordingly, this work evaluated crescent concentrations of tunicamycin (TM µg mL-1): control (0), lower (0.5), mild (1.5), and higher (2.5) on the initial establishment of sorghum seedlings CSF18 and CSF20. ER stress promoted growth and metabolism reductions, mainly in CSF18, from mild to higher TM. The lowest TM increased SbBiP and SbPDI chaperones, as well as SbbZIP60, and SbbIRE1 gene expressions, but mild and higher TM decreased it. However, CSF20 exhibited higher levels of SbBiP and SbbIRE1 transcripts. It corroborated different metabolic profiles among all TM treatments in CSF18 shoots and similarities between profiles of mild and higher TM in CSF18 roots. Conversely, TM profiles of both shoots and roots of CSF20 overlapped, although it was not complete under low TM treatment. Furthermore, ER stress induced an increase of carbohydrates (dihydroxyacetone in shoots, and cellobiose, maltose, ribose, and sucrose in roots), and organic acids (pyruvic acid in shoots, and butyric and succinic acids in roots) in CSF20, which exhibited a higher degree of ER stress tolerance compared to CSF18 with the root being the most affected plant tissue. Thus, our study provides new insights that may help to understand sorghum tolerance and the ER disturbance as significant contributor for stress adaptation and tolerance engineering.