Marked seasonality, especially in sexual reproduction, is common among seaweed species along temperate coasts and increases the possibility of successful fertilization in outcrossing species. A phenological shift in reproductive seasons, therefore, could be an effective isolation barrier between conspecific seasonal populations, although its power has not been verified in algae. <italic>Sargassum horneri</italic>, a major component of seaweed beds along the temperate coast of Japan, is known for variability in its reproductive phenology. To understand the significance of phenological shift as an isolation barrier in seaweed species, phenological investigations of <italic>S. horneri</italic> seasonal populations on the Sea of Japan coast of central Honshu, Japan, were combined with Bayesian cluster analysis based on a nuclear simple sequence repeat genotype. Results from these analyses concordantly suggest a genetic differentiation between the seasonal populations, although almost 20% of field-collected plants were estimated to be hybrids or have a hybrid origin based on results of Bayesian cluster analyses using experimental hybrids. A collapse of seasonal isolation was also detected at the site of the field investigation, and a high percentage of putative hybrids in the following generation at the site (41%) suggested significant seasonal isolation in the differentiation observed in this study.