The anchovies of the genus Lycengraulis are the product of an evolutionary transition from a marine to freshwater environment that occurred in South America during the Miocene epoch. Lycengraulis grossidens originated from freshwater lineages and is currently distributed in estuaries and coastal zones. Nevertheless, based on otolith chemistry, there are landlocked individuals in the Uruguay River. The aim of the present study was to investigate the spatiotemporal scenario by which these landlocked individuals reached their current distribution: whether through a north-to-south continental route based on the connection between basins or from the marine environment. To this end, a fragment of the mitochondrial (mt)DNA control region was analysed from individuals collected along freshwater, estuarine and marine environments. We found a significant genetic differentiation between freshwater and coastal (estuarine and marine) populations. Larger haplotype diversities and female effective population size values were found in the coastal population. Nevertheless, the outgroup rooting positioned some Uruguay River haplotypes as hypothetical ancestors in the directed network and as early offshoots in the phylogeny, suggesting the landlocked population as an ancestral lineage. Therefore, the phylogenetic history and biogeography are consistent with a north-to-south continental colonisation route putatively associated with ancient connections between the Amazon and Parana basins followed by new evolutionary transitions to the coastal region associated with Quaternary sea level oscillations.