Advances in metagenomics have facilitated population studies of associations between microbial compositions and host properties, but strategies to minimize biases in these population analyses are needed. However, the effects of storage conditions, including freezing and preservation buffer, on microbial populations in fecal samples have not been studied sufficiently. In this study, we investigated metagenomic differences between fecal samples stored in different conditions. We collected 46 fecal samples from patients with lung cancer. DNA quality and microbial composition within different storage method were compared throughout 16S rRNA sequencing and post analysis. DNA quality and sequencing results for two storage conditions (freezing and preservation in buffer) did not differ significantly, whereas microbial information was better preserved in buffer than by freezing. In a metagenomic analysis, we observed that the microbial compositional distance was small within the same storage condition. Taxonomic annotation revealed that many microbes differed in abundance between frozen and buffer-preserved feces. In particular, the abundances of Firmicutes and Bacteroidetes varied depending on storage conditions. Microbes belonging to these phyla differed, resulting in biases in population metagenomic analysis. We suggest that a unified storage method is requisite for accurate population metagenomic studies.