Since the application of molecular methods, culture-independent methods (CIMs) have been developed to study microbial communities from various environments. In the past 20 years, several methods based on the direct amplification and analyses of the small subunit ribosomal RNA gene have been developed to directly study environmental microorganisms. These methods include denaturing/temperature gradient gel electrophoresis, single-strand-conformation polymorphism, restriction fragment length polymorphism, terminal restriction fragment length polymorphism, and quantitative polymerase chain reaction (PCR). Similarly, non-PCR-based molecular techniques, such as microarray and fluorescence in situ hybridization have also been adopted. In recent years, several novel fields of investigation such as metagenomics, metatranscriptomics, metaproteomics, and single-cell genomics were developed, largely propelled by the innovation and application of next-generation sequencing methods. Several single-cell-based technologies such as Raman microspectroscopy and nano-scale secondary ion mass spectrometry are also increasingly used in the fields of microbial ecology and environmental microbiology. The application of these methods has revolutionized microbiology by allowing scientists to directly analyze natural microbial communities in situ, including their genes, transcripts, proteins, and metabolites and how their interactions impact their distribution patterns. In this review, we present an up-to-date review on different CIMs and their applications, our focuses are on the comparison of different CIMs and their application in the analyses of microbial diversities and communities.