Heat shock proteins have been shown to play an essential role in protecting organisms from environmental stresses and can be divided into multiple families, including the heat shock protein 70 family. The family is composed of heat shock protein 70 (Hsp70) and heat shock cognate 70 (Hsc70). In the present study, to investigate whether the Hsp70 family plays an important role in the thermotolerance of Bombyx mori (silkworm), we analysed the expression of the Hsp70 family before and during heat shock in the midgut of three strains, including a thermotolerant strain cs-10, a thermosensitive strain cs-11, and cs-7, a thermotolerant near isogenic line of cs-11. qRT-PCR and Northern blot analyses revealed that, before heat shock, there was no significant difference in the expression of the selected four Hsp70 family members (Hsp70B, Hsp68, Hsc70-4 and Hsc70-3) among the three strains. Interestingly, after a heat shock of 2 h at 40 °C, increased expression of the selected four members was observed in all three strains, suggesting they are all inducible and involved in thermotolerance. Remarkably their expression, especially that of Hsp70B and Hsp68, was positively correlated with basal thermotolerance. After a heat shock of 8 h at 40 °C, there was no significant difference in the transcript level of Hsc70-3 between cs-10 and c-11. However, its transcript levels in both strains were still significantly higher than its pre-shock levels, indicating that Hsc70-3 may play an important role in long-term basal thermotolerance by working with other proteins yet to be identified. Unexpectedly, transcript levels of Hsp68, Hsc70-3 and Hsc-4 were found to be higher in cs-7 than in cs-10. A hypothesis is proposed to explain these results.