In order to determine the ecological status of surface waters of different types in a particular ecoregion, the reference conditions for each type should be identified to compare with observed conditions, as suggested by the Water Framework Directive (WFD). In this study, 36 candidate reference river sites of the Sakarya Basin (Türkiye) were sampled three times in 2018, and the biological assessment of these sites was evaluated using 18 different diatom indices. In addition, nine different environmental variables were also sampled combined with epilithic diatoms. Based on the canonical correspondence analysis (CCA), alkalinity and pH were determined as the most significant environmental variables for diatom assemblages. Among the diatom indices, Biological Diatom Index (IBD), Artois‐Picardie Diatom Index (IDAP), Descy's Index (DES) and Sládeček's Index (SLA) showed significant correlations with alkalinity and pH according to multiple linear regression (MLR) results. IBD index showed the highest response to the pressures and indicated high or good ecological status of the sampling sites. Moreover, rivers with high water quality status have a high correlation with alkalinity, pH and pollution‐sensitive species. On the other hand, good water quality rivers were highly correlated with total phosphorus and intermediate pollution‐sensitive or pollution‐tolerant species. The 13 reference sites established in this study can be used to assess the ecological status by comparison with the same types of river bodies for further studies in the basins of Türkiye.