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In the European context, several methodologies have been developed to assess the ecological status and, specifically, the hydromorphological status of running surface waters. Although these methodologies have been widely used, few studies have focused on hydrologically altered water bodies and the factors that may determine their status. In this study, the Serpis River was divided into 16 segments from the Beniarrs dam (40 km from the river mouth) to the sea, all of which are affected by flow regulation, but with different severity. In each segment, an inventory of the flora was conducted, and hydromorphological indices (QBR, Riparian Habitat Quality Index; and IHF, River Habitat Index) were applied. The objectives of the study were (A) to identify relationships between floristic composition and QBR components and (B) between instream habitat characteristics and IHF components as well as (C) to determine the main factors controlling the floristic composition and riparian habitat quality (QBR) and the factors controlling instream habitat characteristics and heterogeneity (IHF). A cluster analysis allowed grouping sites according to their floristic composition and instream habitat characteristics, and non-metric multidimensional scaling (NMDS) was used to ordinate the sites, obtaining the biotic and instream habitat characteristics, as well as the QBR and IHF subindices, which better explained the spatial patterns. Finally, a canonical correspondence analysis (CCA) with predictor variables (geographical, hydrological, geomorphological and human pressures) indicated the main factors controlling the spatial patterns of the floristic composition, instream habitat characteristics, riparian habitat quality and instream habitat heterogeneity. A gradient of riparian and instream habitat quality was identified. Our results suggest that physical constraints (presence of a gorge) have protected sites from severe human impacts, resulting in good ecological quality, despite hydrological alteration. This area, where there is geomorphological control, could be potentially reclassified into a different ecotype because regular monitoring could be using incorrect references for index scores, and naturally high scores could be confused with recovery from hydrological alteration or other pressures. The sites with the worst quality were near the river mouth and were characterised by an artificial and highly variable flow regime (related to large autumnal floods and frequent human-induced periods of zero flow). This artificial flow variability as well as the presence of lateral structures in the river channel and geomorphological characteristics were identified as the main factors driving the hydromorphological and floristic pattern in this regulated river.