Cotton’s susceptibility to low temperatures makes it a crucial raw resource for the world’s textile industry, yet its cultivation in temperate regions is severely limited. Although plant growth and stress responses depend on receptor-like kinases (RLKs), the functions of the MEDOS (MDS) gene family, which includes genes that encode RLK, are still poorly understood in cotton. In this study, we conducted a genome-wide analysis to systematically investigate the distribution of MDS gene family members in four cotton species. Phylogenetic analysis identified five evolutionary clades of the MDS gene family in cotton. The role of promoter cis-acting elements in hormone signaling and abiotic stress responses was suggested by analysis. Collinearity analysis demonstrated that segmental duplication was the primary driver of family expansion. Gene expression profiling showed that GhMDS11 was significantly upregulated under cold stress. Functional validation through silencing GhMDS11 compromised cold tolerance, confirming its role in stress adaptation. Comparative transcriptome study of silenced plants demonstrated substantial enrichment in pathways associated with hormone signal transduction and fatty acid breakdown. It is speculated that the chain of “hormone synthesis → signal transduction → secondary metabolism” completely presents the transcriptional regulation network and functional response of plants after receptor kinase VIGS. Silencing the GhMDS11 gene in cotton initiates regulatory effects through hormone synthesis, which is amplified via a signal transduction cascade, ultimately affecting secondary metabolism. This comprehensive pathway clearly demonstrates the downstream transcriptional reprogramming and functional changes. This work thoroughly examined the evolutionary traits of the MDS family across four cotton species and clarified the functional and molecular processes of GhMDS11 in improving low-temperature tolerance, laying a solid foundation for further clarifying multidimensional regulatory networks and breeding cold-resistant cotton materials. Simultaneously, our findings pave the way for future research to develop molecular markers, which could potentially shorten the breeding cycle and facilitate the targeted enhancement of cold tolerance in cotton.