Brassinosteroids (BRs) are plant steroid hormones and, when applied exogenously, they induce physiological responses, including tolerance to heat shock (HS). How endogenous BR content and altered perception of BRs influence thermal tolerance is poorly understood. BR-induced thermotolerance in tomato seedlings with altered BR homeostasis was examined by assessing the survival, ion leakage and lipid peroxidation of seedlings from a BR-deficient mutant (extreme dwarf d(x)), a partially BR-insensitive mutant curl3(-abs) allele (curl3 altered brassinolide sensitivity) and a line overexpressing the Dwarf, BR-biosynthesis gene (35SD). We confirmed that treatment with 1 μM of epi-brassinolide (EBL) induces thermotolerance of wild type seedlings following a HS regime at 45 °C. The curl3(-abs) seedlings had the highest basal tolerance to heat, whereas the EBL-induced thermal tolerance of d(x) seedlings was greatest and responded to lower EBL concentrations. The d(x) and 35SD seedlings had similar thermal tolerance; however, they showed increased signs of oxidative stress. EBL reduced the induction of lipid peroxidation of seedlings after recovery from heat. Highest oxidative stress and peroxidase (POX) activity (EC 1.11.1.7) was in BR-deficient d(x) mutant seedlings. EBL was able of inducing POX activity but not other antioxidant enzymes; however, effects of HS on POX activity of seedlings were absent or less marked. Taking together, results indicate that thermal tolerance is independent of endogenous BR content, but HS-mediated oxidative stress depends on BR levels.