The Krka River flows into the Adriatic Sea and its estuary is known to have been formed in the early Holocene. Generally, karst freshwater in this region encounters potential salinization, creating a link for the transition of various species from marine to freshwater habitats. Due to the high conductivity in karst freshwater ecosystems, it is assumed that species in these environments could more easily overcome osmotic stress during adaptation to freshwater conditions. In this study, we provide a description of new freshwater species of the predominantly marine genus Berkeleya observed in the Krka River. Berkeleya fluviatilis sp. nov. resembles the brackish/marine species Berkeleya rutilans, making it a potential model for studying time-calibrated evolution into freshwater environments in such areas.