A geochemical approach using stable oxygen isotopes was used to understand streamflow generation processes in the highly peaty catchment of Rokytka Brook in the headwaters of Vltava River, Czech Republic. Contribution of water from peat bog areas to the total surface runoff was assessed using a hydrological time series, as well as geochemical, hydrochemical and isotope-hydrological approaches for unit hydrogram separation by means of anion deficiency. Using data from the hydrological year 2008, the role of an existing peat bog in the runoff formation dynamics of Rokytka Brook catchment was determined, and the hydrological cycle was described and assessed using stable 18O/16O isotopes. The research findings strongly support the fact that peatland areas within the studied catchment do not significantly communicate hydraulically with surface streams, and their hydrological function in this region is insignificant.