This research aimed at developing the Trophic Water Quality Index (TWQI) for subtropical temperate Brazilian lotic systems based on a review of the indicative values of diatom species obtained using multivariate analysis techniques and considering the environmental gradient defined by a series of measured physical, chemical, and microbiological variables. Sampling was conducted from 2005 to 2009 in the Pardo River Hydrographic Basin, Rio Grande do Sul (RS), Brazil, and in the Andreas Stream Hydrographic Basin, RS, from 2012 to 2013. A total of 140 biological samples and 211 abiotic samples were collected. Data were analyzed by cluster analysis based on the Ward method and canonical correspondence analysis (CCA). The results indicated that total phosphate, turbidity, ammonia nitrogen, electrical conductivity, dissolved oxygen, and thermotolerant coliforms showed a significant correlation with the sample ordination made by CCA, in relation to a gradient of eutrophication. Eutrophication was operationally defined in a broad sense, including the problem of organic pollution and eutrophication of the water. The determination of the different tolerance degrees to eutrophication of the diatom taxa was used to assign trophic values of 1, 2.5, and 4 to species, corresponding to levels of low, medium, and high tolerance, respectively. By using the trophic values obtained for each diatom species, the TWQI constituted a new technological tool for environmental monitoring studies and showed a consistent, robust, and objective database for water quality assessment in subtropical temperate Brazilian lotic systems.