The Maroni is one of the most speciose basins of the Guianas and hosts a megadiverse freshwater fish community. Although taxonomical references based on morphological identification exist for both the Surinamese and Guianese parts of the basin, there are still taxonomical uncertainties concerning the status of several species. We used COI sequences of 1,284 fish in conjunction with morphological and biogeographical evidence to assist with species delineation and discovery in order to validate and standardise the current taxonomy. This resulted in a final DNA barcode dataset of 199 fish species (125 genera, 36 families, and 8 orders; 68.86% of strictly freshwater fishes from the basin), among which 25 are new putative candidate species flagged as requiring taxonomic update. DNA barcoding delineation through BINs unveiled further cryptic diversity (230 BINs in total). To explore global genetic patterns across the basin, genetic divergence landscapes were computed for 128 species, showing a global trend of high genetic divergence between the Surinamese south‐west (Tapanahony and Paloemeu), the Guianese south‐east (Marouini, Litany, Tampok…), and the river outlet in the north. This could be explained by lower levels of connectivity between these three main areas and/or the exchange of individuals between these areas and the neighbouring basins. A new method of ordination of genetic landscapes successfully assigned species into cluster groups based on their respective pattern of genetic divergence across the Maroni Basin: genetically homogenous species were effectively discriminated from species showing high spatial genetic fragmentation and possible lower capacity for dispersal.