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In compliance with the European and Italian regulations, the Environmental Protection Agency of Umbria Region (ARPA Umbria) defined specific river monitoring programs and networks based on river type definition, human pressures and risk analysis. The Umbria Region lies in Central Italy and it can be split into three hydro-ecoregions belonging to the Mediterranean area. Data on diatom community composition were collected in five different Mediterranean macrotypes (M1-M5) throughout the diatom-based river monitoring network that is composed by 52 sampling stations in 36 watercourses. The main aim of this study was to characterise and to analyse diatom diversity across the different regional river macrotypes. Specifically, we investigated if: i) there were differences in species diversity (species richness and Shannon Index) among macrotypes; ii) there was difference in three water quality indexes (ICMi, IPS, and TI) among sites; and iii) there was a relationship between the observed ICMi, IPS and TI value and the diatom diversity. Two-hundred diatom species and varieties were identified, and the number of species per sampling station ranged from a minimum of 10 to a maximum of 38 species. The most frequent and abundant species were Amphora pediculus, Achnanthidium minutissimum, Navicula cryptotenella, Nitzschia dissipata, and each macrotype showed some peculiar species. The ecological status evaluation based on Intercalibration Common Metric Index (ICMi) classified 69% of the water bodies in high or good class. Significant differences in diversity and ICMi value among stream macrotypes were found, with M4 (small and medium mountain) and M5 (small, lowland, temporary) typologies showing the lowest species richness, and with M5 showing the lowest Shannon Index. Conversely, M2 (small and medium lowland) and M5 showed the highest ICMi value. Lastly, significant correlations between Shannon Index and the ICMi, IPS and TI indexes were found.