This study presents a comparative analysis of human gut microbiota using barcoded pyrosequencing. The method involves amplifying a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences, enabling the parallel analysis of hundreds of samples with minimal processing. In silico modeling demonstrated that the method accurately describes microbial communities down to phylotypes below the genus level. The technique was applied to analyze microbial communities in throat, stomach, and fecal samples, revealing the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology. The results showed that fecal samples formed a distinct cluster, while throat and stomach samples grouped more closely. The majority of annotated reads belonged to five bacterial phyla: Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Fusobacteria. The throat microbiota displayed the lowest phylotype richness, with 152 phylotypes, of which 20 represented 90% of the reads. The stomach microbiota was diverse, harboring 262 phylotypes representing 13 phyla, including reads from phyla not previously detected in the stomach. The three H. pylori-positive stomach samples were dominated by this bacterium, comprising 93–97% of the reads, indicating its strong adaptation to the stomach environment. The lower intestine was found to be dominated by Actinobacteria, which were more abundant than Bacteroidetes in all six fecal samples analyzed. The study highlights the potential of barcoded pyrosequencing as a powerful tool for analyzing microbial communities in various human habitats. The method provides a means for large-scale, well-designed epidemiological studies to investigate the association between microbial gut ecology and diseases. The results confirm previous cloning-based investigations of the gastrointestinal tract and provide novel insights into the throat microbiota. The study also addresses the effect of sequencing errors on taxonomic classifications, demonstrating that the method is robust despite potential errors. The findings underscore the importance of microbial communities in health maintenance and their potential role in disease development.This study presents a comparative analysis of human gut microbiota using barcoded pyrosequencing. The method involves amplifying a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences, enabling the parallel analysis of hundreds of samples with minimal processing. In silico modeling demonstrated that the method accurately describes microbial communities down to phylotypes below the genus level. The technique was applied to analyze microbial communities in throat, stomach, and fecal samples, revealing the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology. The results showed that fecal samples formed a distinct cluster, while throat and stomach samples grouped more closely. The majority of annotated reads belonged to five bacterial phyla: Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Fusobacteria. The throat microbiota displayed the lowest phylotype richness, with 152 phylotypes, of which 20 represented 90% of the reads. The stomach microbiota was diverse, harboring 262 phylotypes representing 13 phyla, including reads from phyla not previously detected in the stomach. The three H. pylori-positive stomach samples were dominated by this bacterium, comprising 93–97% of the reads, indicating its strong adaptation to the stomach environment. The lower intestine was found to be dominated by Actinobacteria, which were more abundant than Bacteroidetes in all six fecal samples analyzed. The study highlights the potential of barcoded pyrosequencing as a powerful tool for analyzing microbial communities in various human habitats. The method provides a means for large-scale, well-designed epidemiological studies to investigate the association between microbial gut ecology and diseases. The results confirm previous cloning-based investigations of the gastrointestinal tract and provide novel insights into the throat microbiota. The study also addresses the effect of sequencing errors on taxonomic classifications, demonstrating that the method is robust despite potential errors. The findings underscore the importance of microbial communities in health maintenance and their potential role in disease development.