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Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-indepen-dent microbial eukaryote community structures using uni-versal primers remains very difficult due to the tremen-dous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communi-ties compared to what can be achieved with universal primers. We discovered that many phylum- or group-specific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from di-verse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We per-formed an assay using two complementary methods, py-rosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6 % for the clone library and 99.2 % for pyrosequencing, respectively. In par-ticular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data re-vealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library