Metagenomics is providing an unprecedented view of the taxonomic diversity, metabolic potential and ecological role of microbial communities in biomes as diverse as the mammalian gastrointestinal tract, the marine water column and soils. However, we have found a systematic error in metagenomes generated by 454-based pyrosequencing that leads to an overestimation of gene and taxon abundance; between 11% and 35% of sequences in a typical metagenome are artificial replicates. Here we document the error in several published and original datasets and offer a web-based solution (http://microbiomes.msu.edu/replicates) for identifying and removing these artifacts.