Endophytes possess an array of plant growth-promoting (PGP) activities and produce bioactive metabolites that act as valuable sources for agriculture, biomedicine, and several related industries. Woody plants grow in girth by the activity of cambium and host a highly diverse community of endophytes, most of which have not been isolated and characterized, and remain poorly understood as far as their ecological and biotechnological importance is concerned. Limitations associated with conventional culture plate-based methods to identify and characterize microbes based on morphological characteristics lead to underestimations of the microbial diversity. Molecular methods involving 16S ribosomal RNA (rRNA) and Internal Transcribed Spacer (ITS) amplification and sequencing have emerged as more reliable tools for the identification of endophytes within a given sample. Next-generation sequencing (NGS) has allowed the comparison of sample phylogeny and taxonomy even from highly complex microbiomes. 16S rRNA gene, conserved among bacteria with specific variable regions, is used for phylogenetic classification of species in diverse microbiomes. Similarly, ITS rDNA is widely used to identify endophytic fungi and requires DNA isolation, PCR amplification, sequencing, and establishing the phylogenetic affinity. Convenient usage, high specificity, and reproducibility have made this universal barcode marker to be widely used for identification purposes. This chapter gives a comprehensive description of the techniques for the assessment of bacterial and fungal endophytes inhabiting the tissues of woody plants.