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1. The European Water Framework Directive provides a framework for improving the ecological quality of stream ecosystems, with deviation from reference used as a measure of ecological status. 2. Here we examine the possibility of using less impacted stream sites from Latvia, Lithuania and Poland to establish a Danish reference network for macrophyte assemblages, and as a guiding image for identification of possible references sites within Denmark. Both approaches were evaluated using historical Danish records. 3. Four different macrophyte assemblages were identified for mid-sized streams in the Central and Eastern Lowland ecoregions. Macrophyte assemblages could not be delineated using physical stream site characteristics; however a gradual change in assemblage composition was attributed to differences in alkalinity and human impact. 4. Assemblages of contemporary vegetation in Denmark were quite similar to those found in Polish, Latvian and Lithuanian streams (26–35%). However, more importantly, from species-based predictions we noted higher similarity, particularly with Latvian and Lithuanian streams, before intensive land use commenced in Denmark ( c. 1900). These results show that stream sites from these three countries can be considered in a Danish reference network. 5. Two of the four macrophyte assemblages comprised species such as Fontinalis antipyretica, Myriophyllum spicatum, Nuphar lutea, Potamogeton alpinus and P. perfoliatus that have a very scattered occurrence in the contemporary vegetation in Denmark. These groups were closely associated with the predictions from historic records, thereby lending support the conjecture that these assemblages could be part of the guiding image for the identification of potential reference sites within Denmark. [ABSTRACT FROM AUTHOR]