Δ9 stearoyl ACP desaturase gene (GhSAD2) is a fatty acid synthesis and metabolism in the process of key desaturase gene. To clarify the gene function in biosynthesis of cotton fatty acids, we cloned the GhSAD2 gene from upland cotton, and analyzed the gene sequence features, evolutionary relationship and expression characteristics. In this study, a fulllength cDNA of stearoylACP desaturase gene was cloned from upland cotton ‘Xinluzao 33’ leaves successfully by using electronic stitching and RTPCR, which named GhSAD2 and access number was KX197920 in GenBank. The gene nucleotide and amino acid sequences were analyzed by using NCBI and DNAMAN and a phylogenetic tree was constructed by using Clustalw 1.83 and MAGE 5.0 software, which would help analyze the gene evolutionary relationships among different species. Result showed that the GhSAD2 gene open reading frame had 1188bp, encoding a polypeptide of 396 amino acids. Sequence analysis revealed that this gene GhSAD2 had two highly conserved histidine clusters of ferritinlike superfamily: EENRHG and DEKRH were located at amino acid 185 and 271. Phylogenetic analysis showed that GhSAD2 homologous gene evolutionary relationship with the cacao tree is very close, which is consistent with the status of the species in the classical taxonomy. qPCR showed that GhSAD2 in G. hirsutum had a much higher expression in leaves than that in stems and roots. GhSAD2 express highest at 25 days (s25) after flowering seed. GhSAD2 in leaves expression under cold stress analysis showed that the gene were upregulated under varying low temperature treatments. It has the highest expression at 6 hours treatment, and then gradually reduced. Studies have shown that GhSAD2 gene may play an important role at cotton seed oil unsaturated fatty acid synthesis. At the same time, it has a certain physiological role in the cold resistance of cotton.