Burrowing mayfly nymphs (Ephemeroptera) inhabit and ingest fine-grained sediments and detritus that may be enriched with metals and persistent organic compounds. The burrowing nymphs can externally adsorb and internally assimilate these contaminants, providing a link for the food chain transfer of potentially toxic substances from sediments to organisms in higher trophic levels. The emergent adults are short-lived and do not feed, thus their gut contents do not contribute greatly to their total contaminant burden. These characteristics make Hexagenia spp. and certain other burrowing mayflies useful for assessing ecosystem contamination. General protocols are presented for the collection, processing and analysis of emergent mayflies to assess the spatial distribution and bioaccumulation of sediment-associated contaminants in aquatic ecosystems. Two essential components of this bioassessment approach are a network of on-site volunteers with the materials and instructions needed to correctly collect and store samples and quality assurance procedures to estimate the accuracy of chemical analyses. The utility of this approach is demonstrated with an example of its application to the Upper Mississippi River (USA). Determination of cadmium, mercury and polychlorinated biphenyl congeners in emergent Hexagenia bilineata from a 1250km reach of this river revealed (1) several source areas of contaminants and (2) distinct patterns in the bioaccumulation (and apparent sediment-associated transport) of each residue on both small and large spatial scales.