A typical environmental sample includes hundreds to thousands of organisms and a biomonitoring regime often requires multiple environmental samples that are repeated over time and space. Hence, the bottleneck in this case may not only be at the DNA sequencing step but can also occur at the collection, sorting, and preparation steps. Working with specimens in a one-at-a-time fashion, is tedious, time-consuming, and expensive. Since longer sequence length means better taxonomic resolution, the 454 Genome Sequencer FLX is the preferred NGS platform for biodiversity studies as it is capable of providing 250�C400 base long sequence reads versus less than 100 bases for the two competing platforms. This property is important because DNA fragments that are sequenced in each sequencing reaction will be examined bioinformatically to derive biodiversity measures from a given environmental samples. It has been shown that longer sequences can provide more accurate biodiversity information such as species-level resolution. The majority of biodiversity studies using this equipment have targeted prokaryotic biodiversity in different environmental samples, from the ocean floor to human micro-flora. These studies typically use sequence variation in a short fragment of ribosomal genes for estimating the diversity of bacteria in the sample. The results are compared to a relatively large sequence library of 16S genes using statistical clustering methods such as BLAST. The same approach can be applied to large environmental samples of eukaryotic organisms. It has been shown that a small mini-Efaproxiral Sodium barcode fragment of the mitochondrial cytochrome c oxidase 1 DNA barcode sequences a sequence length that can readily and robustly be obtained through 454 pyrosequencing��can provide the information required for identification of individual species with more than 90% species resolution. Since early 2008, we have started a technology development project to utilize NGS in biomonitoring programs. We established a NGS facility at the Biodiversity Institute of Ontario, aimed at reconstructing the species composition of environmental samples of eukaryotes. Here we present our preliminary work on samples collected at two locations focused on two of the more important freshwater macroinvertebrate groups: caddisflies and mayflies. Next-generation sequencing is increasingly being used in metagenomics studies to determine the occurrence of microbial taxa. For small-sized taxa which are difficult to culture, nextgeneration sequencing technologies have proved useful in revealing their biodiversity, or for the comparative analysis of microbial biota. However, aside from a few studies�� mainly focused on data analysis and sequencing error rates�� next-generation sequencing has not been directly compared to other identification methods especially for eukaryotic biota. Here we designed and executed our experiments to make comparisons between 454 pyrosequencing and traditional Sanger sequencing based DNA barcoding. Our aim has been to evaluate the feasibility of 454 pyrosequencing to overcome two important challenges faced by biodiversity researchers and environmental Sulindac agencies using benthic macroinvertebrates for their studies. The first challenge is sorting and analysing small specimens especially larvae that are typically used in benthic biodiversity analysis�Coneby-one.