Culture-independent methods to detect microorganisms have been developed in parallel with traditional culture-based methods ever since the classification of bacteria based on 16S rRNA gene sequences was advocated in the 1970s. The development and the prevalence of culture-independent molecular technologies have provided revolutionary progress in microbial studies. The development of these technologies contributes significantly to the research of microorganisms that cannot be detected by traditional methods such as culture-dependent methods.Many molecular methods targeting the 16S rRNA gene, such as fluorescence in situ hybridization (FISH), quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), denaturing-gradient gel electrophoresis (DGGE), clone library analysis, and next-generation DNA sequencing (NGS) technologies, have been applied to various microbial studies. Notably, the advent of NGS technologies enabled a large-scale research of the bacterial community. Many recent studies using the NGS technologies have revealed that a larger number of bacteria and taxa than previously thought inhabit various parts of the human body and various places on the earth. The principles and characteristics of each molecular method are different, and each method possesses individual advantages; for example target specificity, comprehensiveness, rapidness, and cost efficiency. Therefore it is important that the methods used in studies are suitable for the objective and materials. Herein, we highlights molecular approaches targeting the 16S rRNA gene in bacterial community analysis, and focuses on the advantages and limitations of each technology.