Benthic diatoms are widely used in Europe and worldwide to access ecological status of running waters. One of key goals of Water Framework Directive is to classify rivers and streams using biological quality elements and type specific reference conditions. According to system B which incorporates additional abiotic descriptors, there are 24 water types in Croatia. For biological analyses 92 rivers and streams with 140 sampling points were chosen and sampled for benthic diatoms and water chemistry simultaneously. Self organizing map (SOM) analysis was used to define biotypes from species composition and abundance of benthic diatoms. Grouping of samples in SOM resulted in 10 distinctive groups. Based on their geographical position and site characteristics, groups represent sites with similar properties (as waterbed, catchment size, altitude, size of stream) belonging to different ecoregions in Croatia. Analysis of variance revealed statistically significant differences (p<0.05) among SOM groups concerning ammonia, nitrates and total phosphorus. Indicator species analysis (IndVal) singled out species that were significantly characteristic (p<0.05) for SOM and abiotic types. Compared to abiotic groups, in which 7 out of 24 have no indicator species, all SOM groups have one or several characteristic diatom species, thus indicating diatom assemblages as valuable site descriptors. Canonical analysis of principal coordinates analysis also indicated that SOM grouping of samples is statistically reliable. Grouping of similar sites, although placed into different abiotic types, makes SOM groups with its corresponding representative species an easy tool for water quality assessment and description of reference assemblage.