Phytoplasmas are plant pathogens transmitted by insects that pose significant challenges for the most common experimental tasks. This is especially true concerning analyses that aim at genome sequencing, due to the phytoplasma’s low abundance and its association with the host cellular matrix. This protocol provides a roadmap for researchers in the field of phytoplasma genomics, focusing on three crucial aspects: (1) DNA extraction from plant hosts, including a protocol for low-concentration samples, (2) Sequencing strategies, emphasizing accurate base calling (particularly for Oxford Nanopore) and addressing mixed plant/phytoplasma samples, (3) Phytoplasma genome assembly and analysis, detailing methodologies and exploring computational/comparative techniques to decode phytoplasma genomes. With a focus on tackling pivotal challenges and harnessing possibilities proposed by these advancements, this approach equips researchers to unlock the secrets of these enigmatic plant pathogens.