In our previous study, we reported that the nucleotide sequence of a 14-3-3ε cDNA clone from Litopenaeus vannamei encodes two transcripts, designated as 14-3-3EL and 14-3-3ES. Based on the retained introns we observed in 14-3-3EL by alternative splicing, we aimed to investigate the genomic structure of this gene. The 14-3-3ε gene, encoded by 2,311 bp of DNA sequence, is interrupted by three introns of 48, 1,124 and 365 bp in size. In addition, using the SWISS-MODEL, the predicted 3D model of 14-3-3EL has twelve α-helices and two β-sheets, whereas 14-3-3ES has only eleven helices in its tertiary structure. Additionally, we generated the 14-3-3EL and 14-3-3ES proteins and measured their thermal stabilities. The results demonstrated that the quantity of the 14-3-3EL protein decreased significantly at all temperatures tested. In contrast, the quantity of the 14-3-3ES protein remained unchanged, suggesting that 143-3-3EL is less stable than 14-3-3ES.