Classification of natural flow regimes of non-perennial rivers and streams (NPRS) is an incipient field of research. NPRS represent approximately 70% of the total Mediterranean rivers and are expected to increase in the next decades as a result of climate change. Due to the ecological importance of NPRS and the need to improve national ecological assessment methods within the scope of the Water Framework Directive (WFD), this paper aims to classify the hydrological regime of 69 non-regulated streams, testing several hydrological indices related to the frequency, duration, timing, and rate of change in periods of flow cessation. Using daily flow records, a total of 315 indices were calculated and their relationships were examined with Principal Component Analysis (PCA) for different thresholds used to define zero-flow condition set at 0, 1, 2, and 5 l/s. Redundancy analysis identified five indices that better describe the patterns of hydrological variability in Mediterranean NPRS: number of days per year without flow, annual percentage of months without flow, mean daily of annual flows, coefficient of variation of Julian date of the annual start of zero flow and annual rise rate. Using these indices, a self-organizing map (SOM) was trained in order to categorize the NPRS into three groups with similar hydrological features. The results of this study provide a statistically-based hydrological classification of NPRS in Mediterranean environments. We expect that this classification will provide useful insights to water authorities to improve the assessments of the ecological status in this set of water bodies.