The important role of the microbiome in the immune system, nutrient digestion, and disease diagnosis and prevention has been well documented in both humans and animals. In poultry, the microbiome community changes between different organs, and the perturbation of gut microbiota can lead to intestinal infections and respiratory diseases, which result in enormous economic losses. The advances in several high-throughput sequencing techniques have generated big sequencing data for microbiome analysis. In this review, we go over the traditional molecular tools used to analyze gut microbiome in poultry, such as T-RFLP, DGGE, and TGGE, clone library sequencing using the Sanger method, the development of next-generation sequencing techniques such as Roche 454, Illumina, and Ion Torrent. We also discuss third-generation sequencing techniques including Pacbio SMRT sequencing and Oxford Nanopore, both of which were developed to generate length reads. Finally, we discuss other omics such as metagenomics, metatranscriptomics, and metaproteomics, which are necessary for studying microbiome functions.