The microalgae Didymosphenia geminata (Lyngbye) Schmidt, commonly known as Didymo or "Rock snot" is an invasive freshwater benthic diatom, native to rivers of the Circumboreal Region of Europe. In Chile, Didymo was first found blooming in the Espolón River (around 44. °S) in Patagonia in 2010. Several years later, Didymo has expanded its range to 3000. km from 38. °S to 53. °S. Genetic information about this alga could improve the early detection and monitoring of this invasive species especially in uncontaminated rivers. Therefore, this information could help managers to make conservation decisions about places threatened by Didymo invasion. Here, a phylogenetic analysis employing two commonly used molecular markers was conducted to identify Chilean Didymo samples from different spatially distant infested rivers. The data presented herein confirm the presence of Didymo in Chile and include the first genetic account of an invasion in South America.