The rapid increase in the aquaculture production of salmonids has been followed by a rise in several diseases. In particular, saprolegniasis can account for at least 10% of the annual economic loss in salmonids. In this study, we investigated the main Saprolegnia species involved in saprolegniosis of salmonids in Chile, and their association with specific developmental stages of the host fish. For this purpose, we studied 244 isolates of Saprolegnia-affected Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and king salmon (Oncorhynchus tshawytscha) from the main salmon farming regions, using a recently developed identification strategy based on molecular taxonomical operational units. We found that the Saprolegnia species associated with diseased salmon were S. australis, S. delica, S. diclina, S. ferax, S. parasitica and two new Saprolegnia species observed during this study. In order to determine whether there were any specific species associations with different stages in the fish life cycle, we applied mosaic plots and correspondence analyses for categorical data. These analyses showed a strong association of S. parasitica with samples from the adult stage of the fish (χ2 = 196.29, p < 0.0001), while the species S. australis, S. diclina and Saprolegnia sp.2 were strongly associated with embryonic stages (eggs or alevins), (χ2 = 196.29, p < 0.0001). This work represents the first detailed molecular characterization of Saprolegnia species involved in saprolegniasis in Chile, and the first study showing specific association of different Saprolegnia species with different stages in the salmonid life cycle.