Author Posting. © The Authors, 2009. This is the author's version of the work. It is posted here by permission of Society for Applied Microbiology and Blackwell Publishing for personal use, not for redistribution. The definitive version was published in Environmental Microbiology 11 (2009): 1292-1302, doi:10.1111/j.1462-2920.2008.01857.x. ; PCR-based surveys of microbial communities commonly use regions of the small subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using OTU- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of ca. 100 base pair (bp), 400bp, and 1000bp from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates, and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias, and mis-priming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities. ; This work was supported by a National Research Council Research Associateship Award and L'Oréal USA Fellowship (J.A.H.), NASA Astrobiology Institute Cooperative Agreement NNA04CC04A (M.L.S.), the Alfred P. Sloan Foundation's ICoMM field project, and a subcontract from the Woods Hole Center for Oceans and Human Health from the National Institutes of Health and the National Science Foundation (NIH/NIEHS 1 P50 ES012742-01 and NSF/OCE 0430724; J.Stegeman, PI to HGM and MLS).