[Objectives] To investigate the effect of low temperature treatment on cotton leaves at the two-leaf stage. [Methods] Two cotton varieties (CN01 and SJB016 (low temperature-tolerant)) were used as the trial materials. They were treated at 25 (CK) and 12 ℃ (low temperature) for 0, 3, 6, 12, 24, 48 and 72 h, respectively. Then, the changes in the contents of MDA, SS and Pro in the cotton leaves were analyzed. Based on the analysis results, RNA-seq verification was performed. [Results] The MDA, SS and Pro contents of CN01 and SJB016 treated at 12 ℃increased compared with those treated at 25 ℃. After low temperature treatment, the increases of MDA and Pro contents were smaller, and the increase of SS content was larger in SJB016 compared with those in CN01. Considering the above-mentioned indices comprehensively, the changes in the cotton leaves that were treated at 12 ℃ for 6 h were most obvious, so RNA-seq was performed on them. Compared to those treated at 25 ℃, the expression of photosynthesis-related genes in CN01 treated at 12 ℃ all up-regulated, and the expression of photosystem II oxygen-evolving complexes (PsdP, PsdQ) in SJB016 treated at 12 ℃ all down-regulated. The number of up-regulated differentially expressed genes related to plant hormones in CN01 and SJB016 was larger than that of down-regulated genes. Among them, the ethylene-responsive transcription factors ERF053, ERF020 and ERF061 up-regulated significantly. [Conclusions] These genes may play an important role in improving the cold resistance of cotton.