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Summary: Leucine‐rich repeat‐receptor‐like kinase (LRR‐RLK) is a large subfamily of plant RLKs; however, its role in cold tolerance is still unknown. A novel cold tolerance LRR‐RLK gene (MtCTLK1) in Medicago truncatula was identified using the transgenic lines overexpressing MtCTLK1 (MtCTLK1‐OE) and mtctlk1 lines with Tnt1 retrotransposon insertion. Compared with the wild‐type, MtCTLK1‐OE lines had increased cold tolerance and mtctlk1 showed decreased cold tolerance. The impaired cold tolerance in mtctlk1 could be complemented by the transgenic expression of MtCTLK1 or its homolog MfCTLK1 from Medicago falcata. Antioxidant enzyme activities and proline accumulation as well as transcript levels of the associated genes were increased in response to cold, with higher levels in MtCTLK1‐OE or lower levels in mtctlk1 lines as compared with wild type. C‐Repeat‐Binding Factors (CBFs) and CBF‐dependent cold‐responsive genes were also induced in response to cold, and higher transcript levels of CBFs and CBF‐dependent cold‐responsive genes were observed in MtCTLK1‐OE lines whereas lower levels in mtctlk1 mutants. The results validate the role of MtCTLK1 or MfCTLK1 in the regulation of cold tolerance through the CBF pathway, antioxidant defense system and proline accumulation. It also provides a valuable gene for the molecular breeding program to improve cold tolerance in crops. Significance Statement: A novel cold tolerance leucine‐rich repeat‐receptor‐like kinase was identified from Medicago truncatula. It confers cold tolerance through regulation on the C‐repeat‐binding factor cold response pathway, antioxidant defense, and proline accumulation. [ABSTRACT FROM AUTHOR]