Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and incompatible with fluorescence microscopy. Here, we describe assembly and operation of the multiplexed-Fission Yeast Lifespan Microdissector (multFYLM), a high-throughput microfluidic device for rapidly acquiring single-cell whole-lifespan imaging. multFYLM captures approximately one thousand rod-shaped fission yeast cells from up to six different genetic backgrounds or treatment regimens. The immobilized cells are fluorescently imaged for over a week, while the progeny cells are removed from the device. The resulting datasets yield high-resolution multi-channel images that record each cell’s replicative lifespan. We anticipate that the multFYLM will be broadly applicable for single-cell whole-lifespan studies in the fission yeast (Schizosaccharomyces pombe) and other symmetrically-dividing unicellular organisms.