In this study, the antistress At1g67520 gene from Arabidopsis thaliana was used as a probe, a RLCK gene of GbRLCK10 was cloned through the in silico cloning and RTPCR. To reveal the expression, regulation and mechanism of RLCK family genes in Gossypium barbadense, we has analyzed the genomic structure and the expression pattern of GbRLCK10 by combining with qRTPCR technology under mRNA organization and different stress inducements. Results show that: (1) the ORF of GbRLCK10 was 1 179 bp, encoding 392 amino acids. GbRLCK10 contained only the Serine/Threonine kinase domain, and belonged to receptorlike cytoplasmic kinase gene family. Phylogenetic analysis showed that GbRLCK10 closed to GaRLCK10 and named as GbRLCK10 (Genebank: 2022184 ). The gene was made up of 5 exons and 4 introns. (2) The GbRLCK10 expression were detected by qRTPCR, the results showed that GbRLCK10 gene is expressed in root, stem and leaf of resistant cultivar ‘Xinhai 21’ and susceptible cultivar ‘Xinhai 14’. With the duration of the treatment of Verticillium dahliae, while the relative expression of GbRLCK10 was significantly lower and the response was later in the susceptible cultivar than that in resistant cultivar. Both NaCl and PEG6000 could induce the expression of GbRLCK10, the expression of GbRLCK10 increased firstly and decreased. After resistant cultivar ‘Xinhai 21’ was treated by NaCl, response time of GbRLCK10 is earlier than PEG6000, but the response to PEG6000 was more intense. Salicylic acid, ethylene, methyl jasmonate and gibberellic acid could also induce the expression of GbRLCK10. After the treatment of salicylic acid, the level of GbRLCK10 expression increased and decreased, then increased again. The expression of GbRLCK10 increased and maintained at a high level when induced by ethylene. However, the expression of GbRLCK10 firstly increased and then decreased when induced by methyl jasmonate. Compared with salicylic acid, ethylene, methyl jasmonate, the gibberellic acid induced expression levels of GbRLCK10 were significantly lower. We suggested that GbRLCK10 may be response to pathogens, NaCl, PEG6000 and hormones stress in Gossypium barbadense L., but its function still need further study.