The Humboldt Current System (HCS) is characterised by latitudinal upwelling differences and interannual El Niño events that bring equatorial warm waters into the system. Within the HCS, the silverside Odontesthes regia is a coastal fish that faces intense harvesting, and physical and oceanographic variability. Here, we evaluated how different processes in the HCS have shaped the spatial and temporal genetic variation of O. regia populations by using 650-bp alignment of the mitochondrial control region and nine microsatellite loci. We found that both DNA markers indicated high genetic diversity, presence of at least two co-distributed genetic groups, and a slight genetic structure shaped by a biogeographic break within the HCS rather than the latitudinal upwelling differences or geographical distances. Demographic history of these groups suggests an allopatric origin and posterior reconnection as the result of historical changes in the HCS. No genetic interannual divergences were found in subsystems with intermittent presence of warm waters, suggesting no significant influence of El Niño on the genetic variation of O. regia from 2013 to 2018. Despite the inherent disturbances of the HCS, past and recent estimates of effective population sizes showed high values in most populations. Our results provide essential information for the management of O. regia in the HCS. Our study also helps to understand how the spatio-temporal dynamics of the HCS influences the genetic variation of coastal marine fishes.