10Conventional morphology-based identification is commonly used for routine assessment of freshwater 11 ecosystems. However, cost and time efficient techniques such as high-throughput sequencing (HTS) based 12 approaches may resolve the constraints encountered in conducting morphology-based surveys. Here, we 13 characterized stream macroinvertebrate species diversity and community composition via metabarcoding and 14 morphological analysis from environmental samples collected from the Shigenobu River Basin in Ehime 15 Prefecture, Japan. We compared diversity metrics and assessed both approaches' ability to evaluate the 16 relationship between macroinvertebrate community and environmental variables. In total, we morphologically 17 identified 45 taxa (3 families, six subfamilies, 31 genera, and five species) from 8,276 collected individuals 18 from ten study sites. We detected 44 species by metabarcoding, with 35 species collapsed into 11 groups 19 matching the morphologically identified taxa. A significant positive correlation between logged depth (number 20 of HTS reads) and abundance of morphological taxa was observed, which implied that quantitative data can 21 be used for subsequent analyses. Relatively higher estimates of alpha diversity were calculated from the 22 metabarcoding data in comparison to morphology-based data. However, beta diversity estimates between 23 metabarcoding and morphology data based on both incidence and abundance-based matrices were 24 correlated proving that community differences between sampling sites were preserved in the molecular data. 25 Also, both models were significant, but metabarcoding data (93%) explained a relatively higher percentage of 26 variation in the relationship between community composition and the environmental variables than 27 morphological data (91%). Overall, we present both the feasibility and limitations of HTS-driven estimations 28 of taxonomic richness, community composition, and diversity metrics, and that metabarcoding was proven 29 comparable and more sensitive against morphology-based analysis for stream macroinvertebrate biodiversity 30 assessment and environmental monitoring. 31 32 community composition 33 42 al., 2016). 43 Conventional morphological analysis is most commonly used in routine monitoring programs evaluating 44 environmental quality changes. However, this is not only time consuming but has serious issues with accuracy, 45 and consistency in the level of taxonomic identification that highly depends on taxonomic expertise (Hajibabaei 46 et al., 2011). Specifically, small organisms such as the larval stages of stream macroinvertebrates frequently 47 used for river biomonitoring are often difficult or impossible to identify at finer taxonomic resolution (e.g., 48 species level) (Sweeny et al., 2011). A promising alternative approach is DNA metabarcoding -a combination 49 of amplicon-based high-throughput sequencing (HTS) analysis and DNA taxonomy (Hebert et al., 2003). High-50 throughput amplicon sequencing can process large number ...