Genetic diversity is the most basal level of biodiversity and determines the evolutionary capacity of species to adapt to changing environments, yet it is typically neglected in routine biomonitoring and stressor impact assessment. For a comprehensive analysis of stressor impacts on genetic diversity, it is necessary to assess genetic variants simultaneously in many individuals and species. Such an assessment is not as straightforward and usually limited to one or few focal species. However, nowadays species diversity can be assessed by analysing thousands of individuals of a community simultaneously with DNA metabarcoding. Recent bioinformatic advances also allow for the extraction of exact sequence variants (ESVs or haplotypes) in addition to Operational Taxonomic Units (OTUs). By using this new capability, we here evaluated if the analysis of intraspecific mitochondrial diversity in addition to species diversity can provide insights into responses of stream macrozoobenthic communities to environmental stressors. For this purpose, we analysed macroinvertebrate bulk samples of three German river systems with different stressor levels using DNA metabarcoding. While OTU and haplotype number were negatively correlated with stressor impact, this association was not as clear when studying haplotype diversity across all taxa. However, stressor responses were found for sensitive EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa and those exceedingly resistant to organic stress. An increase in haplotype number per OTU and haplotype diversity of sensitive taxa was observed with an increase in ecosystem quality and stability, while the opposite pattern was detected for pollution resistant taxa. However, this pattern was less prominent than expected based on the strong differences in stressor intensity between sites. To compare genetic diversity among communities in river systems, we focussed on OTUs, which were present in all systems. As OTU composition differed strongly between rivers, this led to the exclusion of a high number of OTUs, especially in diverse river systems of good quality, which potentially diminished the increase in intraspecific diversity. To better understand responses of intraspecific genetic diversity to environmental stressors, for example in river ecosystems, it would be important to increase OTU overlap between compared sites, e.g. by sampling a narrower stressor gradient, and to perform calibrated studies controlling for the number of individuals and their haplotypes. However, this pioneer study shows that the extraction of haplotypes from DNA metabarcoding datasets is a promising source of information to simultaneously assess intraspecific diversity changes in response to environmental impacts for a metacommunity.