Mycoheterotrophic plants (MH) are achlorophyllous, do not photosynthesize, and are totally dependent on mycorrhizal fungi for their nutrition. These plants are usually small and underground, only visible above ground level in their reproductive stages, which last for a few days or months in the year. In many tropical regions, MH plants are highly endangered by habitat changes that can make essential fungi unavailable. The research focusing on these plants is still scarce; one probable cause is the methodological difficulties encountered. Our research group is currently studying three species: the orchids Pogoniopsis schenckii and Wullschlaegelia aphylla, and Voyria aphylla (Gentianaceae). In these species, we came across methodological challenges, such as DNA extraction and identification of fungi by non-amplification methods, carried out by fungi isolation in growth culture media. MH plants from tropical regions have a wide range of endophytic fungal species, and some of these grow considerably slowly, making these experiments and analyses difficult. Confocal microscopy images and identification of fungi with specific reagents require proper sample preparation. This Methods Collection aims to include techniques for the identification of some MH species in the field, investigation of pollinating agents and seed dispersers, methods of collecting samples and processing for structural analyses (optical, electronic, and confocal microscopy), isolation and identification of fungi present in different plant organs, carbon cycling, stable isotopes, metagenomics, plastid sequencing, gene loss, genome evolution, and symbiotic germination.