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Understanding the population dynamics of invasive processes has become a pressing concern in a highly connected world faced with ongoing climate change and increased exotic species introduction. In Chile, the invasive freshwater benthic diatom Didymosphenia geminata (Lyngbye) Schmidt has become widespread, expanding across multiple river basins spanning over 3000 km in three years. Here we evaluate the phylogenetic and phylogeographic relationships of D. geminata samples collected throughout the invaded range in Chile, using specific rbcL gene sequence previously published. Genetic sequences for this marker were generated for 19 sample sites, and were compared with available freshwater diatom sequences, as well as with previously published rbcL gene sequences for D. geminata. We find that all genetic sequences collected within Chile present phylogenetic divergences from D. geminata samples collected in Siberia, as well as from samples of the genera Gomphonema, Cymbella and Encyonema. Thus, we validate the invasion by D. geminata, in agreement with existing morphological taxonomic criteria. In addition, a haplotype analysis showed a total of 13 haplotypes, two of which (halpotypes I and IX) found in 12 and 3 populations respectively, while each of the remaining haplotypes presents a single population. Thus, these results are consistent either with the introduction of multiple lineages, or with a rapid genetic differentiation in this invading freshwater diatom. Further genetic sampling, both within Chile and in countries that may have been potential sources of the invasion are needed to test these alternative hypotheses.