Three heat shock protein 70 (Hsp70) cDNAs were isolated from the carmine spider mite, Tetranychus cinnabarinus. They were tentatively named as TCHsp70-1, TCHsp70-2 and TCHsp70-3. Structural analyses showed that all of the three TCHsp70 cDNAs held the full open reading frame (ORF). Putative protein sequences and a phylogenetic tree suggested that TCHsp70-1 and TCHsp70-3 were cytoplasm HSP70 and TCHsp70-2 was endoplasmic reticulum HSP70. Comparison of deduced amino acid sequences of TCHsp70-1 and TCHsp70-3 showed 84.78% identity, TCHsp70-1 and TCHsp70-2 showed 57.33% identity, TCHsp70-2 and TCHsp70-3 showed 58.26% identity. Real-time comparative quantitative PCR revealed that the relative expression of TCHsp70-2 was lower than TCHsp70-1 and TCHsp70-3 at each temperature tested. TCHsp70-1 and TCHsp70-3 shared a similar expression pattern after cold and heat shock compared with their expression at normal temperature (26 °C), but the mRNA expression of TCHsp70-1 was significantly higher and lower than that of TCHsp70-3 at cold and heat shock temperatures (except for 34 °C), respectively. This result possibly indicated the expression patterns of TCHsp70 were affected by their location in different cellular compartments. The results also indicated that three TCHsp70s, especially TCHsp70-1 and TCHsp70-3, may play an important role in mediating tolerance to cold, thermal stress for Tetranychus cinnabarinus.