This account reports a journey of ca. 500 km from the city of Inírida to the Colombian basins of the Cuiarí and Isana Rivers (Guianía department) in April–May 2014, the first botanical expedition to this region. This area exemplifies one of the last innermost and botanically unexplored regions of the northwestern corner of the Amazon basin (the upper Río Negro basin), aside from the fieldwork started some 234 years ago with the exploration of the middle and lower portion of the Isana basin in Brazil (where it is called Içana) by the Portuguese naturalist Alexandre Rodrigues Ferreira. On our expedition we collected 1301 botanical numbers, which resulted in eight new species (two already published), two new families, and 24 species new to the Flora of Colombia. Another noteworthy feature is the high diversity of the forests in the upper Isana River, represented by between 108 and 162 spp. with a diameters ≥ 2.5 cm in different 40 × 40 m plots (1600 m²). The Isana River is a white-water basin (with high levels of sediments), with large areas covered by the vegetation typical of black-water rivers (Amazon Caatinga forests, lower forests, savannas and sclerophyllous shrubby vegetation over white-sand soils). This feature results from the influence of several black-water rivers, the headwaters of which drain from Precambrian Guiana shield outcrops. This account of the first botanical exploration in the Colombian portion of the Cuiarí and Isana Rivers is yet another example of the need for continued floristic studies in regions where there are large geographic gaps in the knowledge of Amazonian flora. Advances in documentation of this flora can be achieved through institutional and private partnerships, improved training, and continued fieldwork in collaboration with local inhabitants. A discussion of demarcation issues regarding the frontier between Brazil and Colombia in this northwestern corner of the Amazon basin, its physical and biological environments, and its ancestral and current inhabitants is presented.