Aureobasidium pullulansis a ubiquitous fungus with a wide variety of morphologies and growth modes including “typical” single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in one cell cycle. The study of A. pullulanspromises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semi-open manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.Movie S1Movie S1Histone H2B‐tdTomato probe shows chromatin condensation during mitosis. H2B‐tdTomato signal in four cells. Inverted‐maximum projection time‐lapse movie at 30 s intervals. Scale bar, 5 μm.Movie S2Movie S2NLS‐tdTomato probe disperses during mitosis. NLS‐tdTomato signal in four cells. Inverted‐maximum projection time‐lapse movie at 30 s intervals. Scale bar, 5 μm.Movie S3Movie S3Sec61‐tdTomato probe reveals nuclear envelope deformation during mitosis. Sec61‐tdTomato signal in four cells. Inverted‐maximum projection time‐lapse movie at 10 s intervals. Scale bar, 5 μm.Movie S4Movie S4Nic96‐tdTomato probe illustrates nuclear pore complex behavior during mitosis. Nic96‐tdTomato signal in four cells. Inverted‐maximum projection time‐lapse movie at 30 s intervals. Scale bar, 5 μm.