Cucumis sativus L.var. xishuangbannanesis is characteristic varieties resource with orange flesh in our country. The beta carotene content has obvious difference between different germplasm. Phytoene synthase (PSY) is the first speed limit enzyme in the pathway of carotene biosynthetic. Full length cDNA and DNA sequences of Psy1 were cloned respectively according to Cucumis sativus L.var.xishuangbannanesis. Corresponding DNA sequence was 2797bp, containing 5 introns and 6 extrons. cDNA sequence was 1385bp, encoding 421 amino acids. The deduced amino acid sequence contains two sequences with high conservation which were characteristic sequences in PSY family. The protein was unstable, having no obvious hydrophobic domain, and the membrane structure was not predicted. The deduced protein PSY1 showed the highest homology with melon, watermelon and bitter gourd based on phylogenetic tree. Four SNPs and one InDel of 13bp were found in Cucumis sativus L.var.xishuangbannanesis by sequence alignment with deep sequencing material "9930" and "GY14". The SNP1 occurred in 27bp of the upstream of the initiation codon, other mutations were located in the introns. SNP1 had the highest specificity based on the sequence analysis of 115 resequencing material. Its mutation rate was 100% in 19 Cucumis sativus L. var. xishuangbannanesis, and 5.3% in 96 cultivated cucumbers. The relative expression of Psy1 in 7 different developmental stages of fruit was studied by real-time fluorescence quantitative PCR. The results showed that the expression level of Psy1 gene increased and then decreased in different cucumber fruit development period, and the amount was significantly different in Cucumis sativus L.var.xishuangbannanesis. In the conversion period (50d after pollination), it reached the maximum value which was more than 8 times of the initial stage of fruit development and more than 4 times of the same stage in common cucumber. The expression amount of common cucumber changed mildly in general. In addition, the amount of expression of the inner peel of Cucumis sativus L.var.xishuangbannanesis was significantly higher than that in the middle peel, and the maximum difference was 5 times. The difference between the inner and middle peel of common cucumber was small relatively. Psy1 may affect the accumulation of beta carotene in Cucumis sativus L.var.xishuangbannanesis.