Scheffersomyces segobiensis DSM27193 has been identified as a potential host for Palmitoleic acid (POA)-rich lipids production. Our previous study has found that S. segobiensis DSM27193 possesses a broad substrate spectrum, excellent xylose fermentation capability, rapid growth, high biomass, and an abundance of intracellular acetyl-CoA. These characteristics make S. segobiensis DSM27193 an ideal chassis for metabolic engineering and the synthesis of various chemicals. However, the genetic coding features of this strain remain unclear. Analysis of the internal transcribed spacer (ITS) region suggested that S. segobiensis DSM27193 is closely related to Pichia stipitis (renamed Scheffersomyces stipitis), both of which exhibit CTG coding characteristic. In this study, we utilized software tools, such as CodonW, CUPS, and others to analyze the codon preference of the S. segobiensis DSM27193 genome. Additionally, we validated the reliability of the codon table through the expression of green fluorescent protein (GFP). The analysis results serve as a theoretical foundation for enhancing the expression of exogenous genes in S. segobiensis DSM27193.