Based on the transcriptome database of Dendrobium huoshanense, we cloned GDP mannose 4,6dehydratase gene (DhGMDS) and the promoter sequence by Genome Walking technique. The tissue expression pattern and expression under low temperature treatment were detected by realtime quantitative PCR, so as to provide a theoretical basis for further analyzing the cold resistance mechanism and genetic improvement of D. huoshanense. Results showed that: (1) DhGMDS gene (GenBank accession number MW855573) was successfully cloned, and its cDNA sequence of the DhGMDS gene was 1 134 bp, and the gDNA sequence was 1 523 bp, without introns. (2) Sequence similarity showed that the identity of DhGMDS protein and GMDS protein of D. catenatum was 99.03%; Phylogenetic tree analysis showed that DhGMDS gene of D. huoshanense and GMDS gene of D. catenatum were in the same evolutionary node, and they had the relatively close genetic relationship. (3) The promoter sequence analysis showed that the upstream promoter region of DhGMDS gene contained light response, low temperature, drought and ABA response elements, MYB transcription factor recognition and binding sites, etc. (4) qRTPCR analysis showed that the expression level of DhGMDS gene was the highest in flowers, followed by stems and leaves, and the lowest in roots. After 72 h of low temperature treatment at 4 ℃, DhGMDS gene was upregulated by low temperature stress, and the relative expression was the highest at 24 h, indicating that the DhGMDS gene was involved in the process of plant stress response. Studies have speculated that the induction of DhGMDS gene expression by low temperature may depend on the regulation of the upstream MYB transcription factor.