Abstract A fundamental prerequisite for assessing the current ecological status of streams is the establishment of reference conditions for each stream type that serve as a benchmark. The hydromorphological reference conditions reflect the natural channel behavior, which is extremely variable. Significant parameters of natural channel behavior were determined using a combination of four selected statistical methods: Principal Component Analysis, Agglomerative Hierarchical Clustering, correlation, and regression. Macroscale analyses of data about altitude, stream order, channel slope, valley floor slope, sinuosity, and characteristics of the hydrological regime were conducted for 3197 reaches of major rivers in the Czech Republic with total length of 15,636 km. On the basis of selected significant parameters and their threshold values, channels were classified into groups of river characteristics based on shared behaviors. The channel behavior within these groups was validated using hydromorphological characteristics of natural channels determined during field research at reference sites. Classification of channels into groups confirmed the fundamental differences between channel behavior under conditions of the Hercynian System and the flysch belt of the Western Carpathians in the Czech Republic and determined a specific group in the flattened high areas of mountains in the Bohemian Massif. Validating confirmed the distinctions between groups of river characteristics and the uniqueness of each one; it also emphasized the benefits of using qualitative data and riparian zone characteristics for describing channel behavior. Channel slope, entrenchment ratio, bed structure, and d 50 were determined as quantitative characteristics of natural channel behavior.