Complementation of auxotrophic nutrient deficiencies in minimal media is widely used for selection of exogenous gene introduction to fission yeast. However, only a limited number of such selection markers are available. Antibiotic resistance markers are good alternatives, but they typically work well in complete rich medium but not in minimal defined Edinburgh minimal medium (EMM). It would be ideal if both the auxotrophic and antibiotic resistance markers can be used together for molecular genetic analysis. Here we describe the use of Zeocin in Pombe minimal glutamate (PMG) media for selection and maintenance of bleMX6 resistance with a LEU2 auxotrophic marker in fission yeast.