Homegardens are recognized worldwide as sustainable agroforestry systems that are repositories of species and genetic diversity. Species diversity and composition of homegardens are influenced by a number of environmental, socioeconomic, and cultural factors. The present study examines the tree species diversity in the homegardens of different cultural groups of Barak Valley, Assam, Northeast India. Complete enumeration was done for diversity analysis in 181 homegardens from 38 villages in the three districts of the Valley. Factors affecting plant diversity were analyzed, and the floristic composition of the homegardens was found to vary with garden size, ethnicity of the homegarden owners, and geographical parameters such as location and altitude. Results indicate high plant diversity, with a total of 161 tree species identified from 47 families. Tea garden labor communities were comparatively found to hold more diversity in their homegardens. Similarity and cluster analysis revealed the distinctness of the majority of the homegardens, and seven groups of homegardens with very low distance were revealed. Homegardens were also found to maintain considerable conservation value as repositories of many underutilized, wild, and rare plant species. Biodiversity conservation in the homegardens was found to be linked to the multiple values of the different plant species to the homegardeners and further highlight the concept of the “conservation through use” approach. For the preservation of such traditional agroforestry systems, which also have the potential to provide options for adaptation to climate change, proper documentation of traditional knowledge concerning the different plant species and the human-nature interaction in the homegardens is necessary before such systems are completely lost.