•IRE is generally considered to be a non-thermal ablation modality.•Treatments with 1–8 µs pulses demonstrate a significant temperature dependence.•Significantly larger ablations were produced at physiological temperatures.•Clinically relevant treatment zones possible with microsecond duration pulses. Irreversible electroporation (IRE) is generally considered to be a non-thermal ablation modality. This study was designed to examine the relative effect of temperature on IRE ablation sizes for equivalent dose treatments with constitutive pulses between 1 and 100 µs. 3D in-vitro brain tumor models maintained at 10 °C, 20 °C, 30 °C, or 37 °C were exposed to 500 V treatments using a temperature control algorithm to limit temperature increases to 5 °C. Treatments consisted of integrated energized times (doses) of 0.01 or 0.1 s. Pulse width, electrical dose, and initial temperature were all found to significantly affect the size of ablations and the resulting lethal electric field strength. The smallest ablations were created at 10 °C and ELethal were calculated to be 1729, 1359, 929, 777, 483 V/cm for 0.01 s treatments with 1, 2, 4, 8, and 100 µs pulses, respectively. At 37 °C these values decreased to 773, 614, 507, 462, and 394 V/cm, respectively. Increasing the dose from 0.01 to 0.1 s at 37 °C resulted in statistically significant decreases (p < 0.001) in ELethal for all treatments except for the 100 µs group. This study found that IRE is a thermally mediated, dose-dependent ablation modality for pulses on the order of one microsecond. Tissue temperatures are not accounted for when determining ablative boundaries in treatment planning algorithms. This work demonstrates that data generated at room temperature may not be predictive of ablation volumes in-vivo and that local temperatures should be accounted for in treatment planning.