Biophysical studies of the mechanochemical cycle of kinesin motors are essential for understanding the mechanism of energy conversion. Here, we report a systematic study of the impact of temperature on velocity and run length of homodimeric Drosophila kinesin-1, homodimeric C. elegans OSM-3 and heterodimeric C. elegans kinesin-II motor proteins using in vitro single-molecule motility assays. Under saturated ATP conditions, kinesin-1 and OSM-3 are fast and processive motors compared to kinesin-II. From in vitro motility assays employing single-molecule fluorescence microscopy, we extracted single-motor velocities and run lengths in a temperature range from 15 degrees C to 35 degrees C. Both parameters showed a non-Arrhenius temperature dependence for all three motors, which could be quantitatively modeled using a simplified, two-state kinetic model of the mechanochemistry of the three motors, providing new insights in the temperature dependence of their mechanochemistry. (C) 2020 The Authors. Published by Elsevier Inc.