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Abstract Background Hoplobatrachus rugulosus (Anura: Dicroglossidae) is distributed in China and Thailand and the former can survive substantially lower temperatures than the latter. The mitochondrial genomes of the two subspecies also differ: Chinese tiger frogs (CT frogs) display two identical ND5 genes whereas Thai tiger frogs (TT frogs) have two different ND5 genes. Metabolism of ectotherms is very sensitive to temperature change and different organs have different demands on energy metabolism at low temperatures. Therefore, we conducted studies to understand: (1) the differences in mitochondrial gene expression of tiger frogs from China (CT frogs) versus Thailand (TT frogs); (2) the differences in mitochondrial gene expression of tiger frogs (CT and TT frogs) under short term 24 h hypothermia exposure at 25 °C and 8 °C; (3) the differences in mitochondrial gene expression in three organs (brain, liver and kidney) of CT and TT frogs. Results Utilizing RT-qPCR and comparing control groups at 25 °C with low temperature groups at 8 °C, we came to the following results. (1) At the same temperature, mitochondrial gene expression was significantly different in two subspecies. The transcript levels of two identical ND5 of CT frogs were observed to decrease significantly at low temperatures (P liver > kidney. Conclusions We mainly drew the following conclusions: (1) The differences in the structure and expression of the ND5 gene between CT and TT frogs could result in the different tolerances to low temperature stress. (2) At low temperatures, the transcript levels of most of mitochondrial protein-encoding genes were down-regulated, which could have a significant effect in reducing metabolic rate and supporting long term survival at low temperatures. (3) The expression pattern of mitochondrial genes in different organs was related to mitochondrial activity and mtDNA replication in different organs.