A series of fission yeast targeting vectors that can be used for wild-type strains having no selectable markers have been designed. The functions of one of three marker genes, lys1(+), arg1(+), and his3(+), involved in amino acid synthesis, are impaired by integration of the fragments generated by restriction enzyme digestion of the plasmids. Successful integration of the fragments into the targeted loci can be readily verified by their requirement for amino acids, or by the PCR diagnostic analysis. Since these selection markers are not used commonly in fission yeast, these plasmids are likely to facilitate studies that require the co-expression of genes such as co-localization and co-immunoprecipitation experiments, by employing them in combination with most of the previously reported markers.