The original yeast strain, Hansenula anomala 2340, was implanted by low-energy nitrogen ion and the mutant library was obtained. Thin layer chromatography (TLC) was used to detect the newly generated red substance produced by mutant strain N6076 and to evaluate the change of metabolic pathways. In order to explore the change in metabolic pathways of mutant strain, a suppression subtractive hybridization (SSH) technology was used to construct a forward subtractive cDNA library of mutant strain N6076. Through sequencing analysis, a total of 14 gene fragment sequences with high expression levels were obtained, which included 9 differentially expressed genes. Among these genes, 7 differentially expressed genes revealed the mutation in bases. Meanwhile, among these 7 differentially expressed genes, 4 genes with base mutation resulted in the change of protein sequences. In addition, the base deletion was also observed in one gene fragment sequence.