Hydrological classification constitutes the first step of a new holistic framework for developing regional environmental flow criteria: the "Ecological Limits of Hydrologic Alteration (ELOHA)". The aim of this study was to develop a classification for 390 stream sections of the Segura River Basin based on 73 hydrological indices that characterize their natural flow regimes. The hydrological indices were calculated with 25 years of natural monthly flows (1980/81-2005/06) derived from a rainfall-runoff model developed by the Spanish Ministry of Environment and Public Works. These indices included, at a monthly or annual basis, measures of duration of droughts and central tendency and dispersion of flow magnitude (average, low and high flow conditions). Principal Component Analysis (PCA) indicated high redundancy among most hydrological indices, as well as two gradients: flow magnitude for mainstream rivers and temporal variability for tributary streams. A classification with eight flow-regime classes was chosen as the most easily interpretable in the Segura River Basin, which was supported by ANOSIM analyses. These classes can be simplified in 4 broader groups, with different seasonal discharge pattern: large rivers, perennial stable streams, perennial seasonal streams and intermittent and ephemeral streams. They showed a high degree of spatial cohesion, following a gradient associated with climatic aridity from NW to SE, and were well defined in terms of the fundamental variables in Mediterranean streams: magnitude and temporal variability of flows. Therefore, this classification is a fundamental tool to support water management and planning in the Segura River Basin. Future research will allow us to study the flow alteration-ecological response relationship for each river type, and set the basis to design scientifically credible environmental flows following the ELOHA framework.