Four vectors were constructed for high-level expression of heterologous proteins with high copy number and mitotic stability in Hansenula polymorpha. All of them contained the conserved H. polymorpha-derived ribosomal DNA (rDNA) sequence for targeting and the geneticin (G418) resistance gene as a selection marker. A strong inducible promoter, formate dehydrogenase (FMD) promoter from H. polymorpha, was used to drive the expression of heterologous genes; the formate dehydrogenase terminator of H. polymorpha was used as the transcription termination region. A modified green fluorescent protein (mGFP) and firefly luciferase protein (Luc) were used as the marker to evaluate the efficacy of these vectors. Using Southern blotting analysis, 2-30 copies of these vectors were integrated into rDNA loci. These results demonstrated that all the four vectors could be used as candidates for expression of desired proteins in H. polymorpha.