Heat shock proteins (Hsps) are stress-responsive molecular chaperones, which uphold proper protein folding in response to external and internal stresses. The Hsp100 gene family plays a substantial role in thermos-tolerance of plants. This study investigated evolutionary relationship and expression of ClpB/Hsp100 gene family in tomato under heat stress. Six SlHsp100 genes were identified using bioinformatics tools. In silico sub-cellular localization indicated that of these 6 ClpB/Hsp100 members, 4 are found in chloroplast, 1 in mitochondria and 1 in the cytoplasm. For evolutionary study, 36 SlHsp100 genes were included in the phylogenetic tree showing a hierarchical clustering shared by the members of the kingdoms Plantae, Archaea, Chromista, Fungi and Bacteria. A total 4 pairs of orthologous and 5 pairs of paralogous genes were identified. Functional divergence between different Hsp100 clusters showed considerable functional homology. Thermo-tolerance measured in terms of cell viability, cell membrane stability and pollen viability indicated that it was paralleled by thermal resistance of Hsps. Reverse transcriptase polymerase chain reaction was used to analyze gene expression in leaves of five-week-old tomato seedlings following exposure to heat stress (45°C) and control (25°C). Chloroplastic LeHSP110/ClpB gene was upregulated in all tomato genotypes after exposure to heat stress highlighting the crucial role of this gene family in acquired thermo-tolerance.