Abstract: In order to study the relevant biological information of the tobacco heat shock protein 70(Hsp70) gene family and the specific expression of the chloroplast-localized members NtHsp70Chl in tobacco midrib, subcellular location, system evolution, Gene structure, chromosome location, and real-time fluorescence quantitative expression PCR analysis were carried out.The results showed that 61 members of the tobacco Hsp70 gene family were identified.The numbers of amino acid contained in the protein sequence were different, and the isoelectric point was in the range of 4.52~9.75.And the protein members were predicted to be localized in the cytoplasm, endoplasmic Reticulum, extracellular matrix, chloroplast (4 members) and mitochondria respectively.The tobacco Hsp70 gene family was divided into 6 groups, and the NtHsp70Chl genes were all in the VI-a subgroup.61 NtHsp70 genes are located on 18 chromosomes, and NtHsp70Chl genes were located on chromosomes 6, 17, and 19, respectively.In tobacco Hsp70 family, there are 10 duplications and 5 paralogous genes.According to RT-qPCR analysis, the NtHsp70Chl genes in tobacco midrib were weakly sensitive to high-temperature stress.This study laid the foundation for further exploration and application of tobacco Hsp70 functions.