Burkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. Burkholderia vietnamiensis LMG10929 and Paraburkholderia kururiensis M130 are bacterial rice growth-promoting models. Besides this common ecological niche, species of the Burkholderia genus are also found as opportunistic human pathogens, while Paraburkholderia species are mostly environmental and plant associated. In this study, we compared the genetic strategies used by B. vietnamiensis and P. kururiensis to colonize two subspecies of their common host, Oryza sativa subsp. japonica (cv. Nipponbare) and O. sativa subsp. indica (cv. IR64). We used high-throughput screening of transposon insertional mutant libraries (Tn-seq) to infer which genetic elements have the highest fitness contribution during root surface colonization at 7 days postinoculation. Overall, we detected twice more genes in B. vietnamiensis involved in rice root colonization than in P. kururiensis, including genes contributing to the tolerance of plant defenses, which suggests a stronger adverse reaction of rice toward B. vietnamiensis than toward P. kururiensis. For both strains, the bacterial fitness depends on a higher number of genes when colonizing indica rice compared to japonica. These divergences in host pressure on bacterial adaptation could be partly linked to the cultivars' differences in nitrogen assimilation. We detected several functions commonly enhancing root colonization in both bacterial strains, e.g., Entner-Doudoroff (ED) glycolysis. Less frequently and more strain specifically, we detected functions limiting root colonization such as biofilm production in B. vietnamiensis and quorum sensing in P. kururiensis. The involvement of genes identified through the Tn-seq procedure as contributing to root colonization, i.e., ED pathway, c-di-GMP cycling, and cobalamin synthesis, was validated by directed mutagenesis and competition with wild-type (WT) strains in rice root colonization assays. IMPORTANCE Burkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. However, the closely related Burkholderia genus includes both beneficial and phytopathogenic species, as well as species able to colonize animal hosts and cause disease in humans. We need to understand to what extent the bacterial strategies used for the different biotic interactions differ depending on the host and if strains with agricultural potential could also pose a threat toward other plant hosts or humans. To start answering these questions, we used in this study transposon sequencing to identify genetic traits in Burkholderia vietnamiensis and Paraburkholderia kururiensis that contribute to the colonization of two different rice varieties. Our results revealed large differences in the fitness gene sets between the two strains and between the host plants, suggesting a strong specificity in each bacterium-plant interaction.