The Harttiini are a tribe of Loricariinae poorly characterized morphologically. Within the Guianas (French Guiana, Suriname, and Guyana), six valid species were recognized, including five Harttia, and the monotypic Harttiella crassicauda. Recent investigations conducted during the last decade by the authors and their co-workers, revealed several unidentified populations of Harttiini. Using a multivariate and multi-table approach unifying morphometry, genetics through DNA barcodes, and ecology-distribution of all populations and species, the global diversity and main evolutionary trends of this group were assessed. The separate analyses highlighted strong structures supporting the validity of three genera: Harttiella, Harttia, and Cteniloricaria, as well as nine new species (six Harttiella, two Harttia, and one Cteniloricaria), and one synonym. The combined analysis established a compromise between the preliminary ones, and revealed their common structure. This structure was found to be linked to the evolutionary history of Harttiini. Their evolution was driven toward adaptations to a definite type of biotope. These included modifications in size and shape, particularly of the caudal peduncle, depending on whether the species colonised rivers or mountainous forest creeks. A longitudinal evolutionary gradient was also highlighted in the geographical distribution of the species despite large overlaps. Notably, Harttiella possessed the greatest number of species with the smallest distribution, making each of them highly vulnerable to anthropic perturbations of their environment. Nine new species are described and a key to all species from the Guianas is proposed.