The analysis of temporal genetic variability is an essential yet largely neglected tool to unveil and predict the dynamics of introduced species. We here describe the temporal genetic structure and diversity over time of an introduced population of the ascidian Styela plicata (Lesueur, 1823) in Wilmington (North Carolina, USA, 34°08′24″N, 77°51′44″W). This population suffers important salinity and temperature changes, and in June every year we observed massive die-offs, leaving free substratum that was recolonized within a month. We sampled 12–14 individuals of S. plicata every 2 months from 2007 to 2009 (N = 196) and analyzed a mitochondrial marker (the gene cytochrome oxidase subunit I, COI) and seven nuclear microsatellites. Population genetic analyses showed similar results for both types of markers and revealed that most of the genetic variation was found within time periods. However, analyses conducted with microsatellite loci also showed weak but significant differences among time periods. Specifically, in the samplings after die-off episodes (August–November 2007 and 2008) the genetic diversity increased, the inbreeding coefficient showed prominent drops, and there was a net gain of alleles in the microsatellite loci. Taken together, our results suggest that recruits arriving from neighboring populations quickly occupied the newly available space, bringing new alleles with them. However, other shifts in genetic diversity and allele loss and gain episodes were observed in December–January and February–March 2008, respectively, and were apparently independent of die-off events. Overall, our results indicate that the investigated population is stable over time and relies on a periodic arrival of larvae from other populations, maintaining high genetic diversity and a complex interplay of allele gains and losses.