The microbial community that live inside plant tissues are known as “endophytes.” The role of endophytes in plant development and plant health stresses the importance of studying this microbial population by means of classic microbiological and culture methods. Low diversity detection is an important problem associated with these methods because the majority of microorganisms are not cultivable. Recent studies of endophytes in roots have focused in understanding diversity, abundance, and distribution of microorganisms in plants using techniques that do not rely on our ability to culture them, such as next-generation sequencing. The analysis of root endophytes using next-generation sequencing depends on correct sampling, on correct storage of roots and on having adequate quality and quantity of metagenomic DNA. Plant tissues contain different compounds that interfere with DNA isolation, such as pigments and phenolic compounds, signifying the importance of correct DNA isolation methods in plant microbiome studies. Here, we describe two different DNA isolation protocols that allow for efficient isolation of metagenomic DNA.