At high latitudes, the polar night poses a great challenge to photosynthetic organisms that must survive up to six months without light. Numerous studies have already shed light on the physiological changes involved in the acclimation of microalgae to prolonged darkness and subsequent re-illumination. However, these studies have never considered inter-individual variability because they have mainly been conducted with bulk measurements. On the other hand, such long periods are likely to impact within-population selection processes. In this study, we hypothesized that distinct subpopulations with specific traits may emerge during acclimation of a population of diatoms to darkness. We addressed this hypothesis using flow cytometry, which allow to individually characterize large numbers of cells. The ecologically dominant polar pennate diatom Fragilariopsis cylindrus was subjected to three dark acclimation experiments of one, three, and five months duration, during which all cultures showed signs of recovery once light became available again. Our results suggest that darkness survival of F. cylindrus relies on reduction of metabolic activity and consumption of carbon reserves. In addition, flow cytometry allowed us to record three different causes of death, each shared by significant numbers of individuals. The first rendered cells unable to survive the stress caused by the return to light, probably due to a lack of sufficient photoprotective defenses. The other two were observed in two subpopulations of cells whose physiological state deviated from the original population. The data suggest that starvation and failure to maintain dormancy were the cause of cell mortality in these two subpopulations.