The biotechnological potential of C. guilliermondii is now well established. This yeast species currently benefits from the availability of a convenient molecular toolbox including recipient strains, selectable markers and optimized transformation protocols. However, the number of expression systems for biotechnological applications in C. guilliermondii remains limited. We have therefore developed and characterized a new series of versatile controllable expression vectors for this yeast. While previous studies firmly demonstrated that knock-out systems represent efficient genetic strategies to interrupt yeast biochemical pathways at a specific step in C. guilliermondii, the set of expression plasmids described in this study will provide new powerful opportunities to boost homologous or heterologous biosynthetic routes by fine controlled over-expression approaches.