This study describes the first detailed molecular characterization of the heat shock protein 70 (Hsp70) gene from Sorghum bicolor, MN1618 designated as SbHsp70-1. The full-length cDNA of SbHsp70-1 consists of 2524 bp with a 1950 bp open reading frame, which encodes a protein of 649 amino acids. SbHsp70-1 is a cytoplasmic protein with high homology to other plant Hsp70s, especially grain crops. Recombinant SbHsp70-1 was able to bind and hydrolyse ATP in a dose-dependent manner, suggesting that SbHsp70-1 functions as an ATPase. Immunoblot assays showed that the expression of SbHsp70-1 is induced at temperatures of 37, 45, and 4 °C but reduced at 42 °C. In addition, the SbHsp70-1 mRNA transcript is constitutively expressed in both leaves and stem but is significantly increased upon heat shock at 42 °C. Upon cold shock at 4 °C, SbHsp70-1 mRNA transcript level increased in the leaf, but no significant change was observed in the stem. In addition, expression of the pET28a-SbHsp70-1 construct in Escherichia coli cells under heat stress resulted in their survival even at higher temperature (65 °C). Our results suggest that SbHsp70-1 is a heat-inducible protein that confer thermal tolerance to bacterial cells and can be claimed as a promising target to study stress tolerance in crops.