The expression of heterologous genes is an important technique in yeast genetics. In fission yeast, theleu1andura4genes have been used mainly as selectable markers for heterologous expression. To expand the repertoire of selection markers available for heterologous expression of genes, here we developed new host-vector systems employinglys1andarg3. By employing genome editing with the CRISPR/Cas9 system, we isolated several alleles oflys1andarg3, each having a critical mutation in the ORF region. In parallel, we developed a set of vectors that complement the amino acid auxotrophy oflys1andarg3mutants when integrated into each locus. Using these vectors in combination with the previously developed integration vector pDUAL, we successfully observed the localization of three proteins in a cell simultaneously by fusing them with different fluorescent proteins. Thus, these vectors enable combinatorial expression of heterologous genes, which addresses increasingly diverse experimental challenges.