Study of the human microbiota in relation to human health and disease is a rapidly expanding field. To fully understand this complex relationship using low-coverage Illumina short reads, we develop a profiling method, microbial nucleotide signatures (MNSs), to characterize the human microbiota. By calculating the nucleotide diversities along the sequenced 16S rRNA gene region, which did not require assembly or phylogenetic identification, we were able to differentiate the gut MNSs of 9 healthy individuals. Applying MNSs to a public dataset showed that it could differentiate body site differences. The scalability of our approach offers a rapid classification of study participants for studies with the sample sizes required for epidemiological studies. Using MNSs to classify the microbiome associated with a disease state followed by a targeted in-depth sequencing will give a comprehensive understanding of the role of the microbiome in human health.