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Sub-optimal temperatures can adversely affect tomato (<i>Solanum lycopersicum</i>) growth, and K⁺ plays an important role in the cold tolerance of plants. However, gene expression and K⁺ uptake in tomato in response to sub-optimal temperatures are still not very clear. To address these questions, one cold-tolerant tomato cultivar, Dongnong 722 (T722), and one cold-sensitive cultivar, Dongnong 708 (S708), were exposed to sub-optimal (15/10 &#176;C) and normal temperatures (25/18 &#176;C), and the differences in growth, K⁺ uptake characteristics and global gene expressions were investigated. The results showed that compared to S708, T722 exhibited lower reduction in plant growth rate, the whole plant K⁺ amount and K⁺ net uptake rate, and T722 also had higher peroxidase activity and lower K⁺ efflux rate under sub-optimal temperature conditions. RNA-seq analysis showed that a total of 1476 and 2188 differentially expressed genes (DEGs) responding to sub-optimal temperature were identified in S708 and T722 roots, respectively. Functional classification revealed that most DEGs were involved in &#8220;plant hormone signal transduction&#8221;, &#8220;phenylpropanoid biosynthesis&#8221;, &#8220;sulfur metabolism&#8221; and &#8220;cytochrome P450&#8221;. The genes that were significantly up-regulated only in T722 were involved in the &#8220;phenylpropanoid biosynthesis&#8221; and &#8220;plant hormone signal transduction&#8221; pathways. Moreover, we also found that sub-optimal temperature inhibited the expression of gene coding for K⁺ transporter SIHAK5 in both cultivars, but decreased the expression of gene coding for K⁺ channel AKT1 only in S708. Overall, our results revealed the cold response genes in tomato roots, and provided a foundation for further investigation of mechanism involved in K⁺ uptake in tomato under sub-optimal temperatures.