Karst areas have some of the most complex aquifers in the world. Because of extreme groundwater oscillations, different hydrological connections are active depending on the season. Therefore, the complex water network remains poorly investigated, despite many geological, hydrological and geomorphological studies performed. The methodology of water tracing was developed especially to determine water interconnections in karst. In addition to tracing with salt, dye or temperature, the use of rich endemic fauna, their dispersion and population structure is lately considered. In this paper, we used population structure of endemic fish species Delminichthys adspersus to confirm hydrological data as well as suggest new underground water connections in part of central Dinaric karst. Moreover, we propose a holistic approach of karst area studies, combining hydrology with biology to develop a better strategy for the protection of vulnerable karst ecosystems. Copyright © 2011 John Wiley & Sons, Ltd.