Abstract Conservation actions for threatened species and populations aim to preserve their genetic diversity and prevent issues such as inbreeding or outbreeding depressions. This perspective has been incorporated into the management plans proposed for the critically endangered Orinoco crocodile (Crocodylus intermedius) in Colombia. A previous study, which used mitochondrial cytochrome b and cytochrome c oxidase subunit I, suggested that the in situ population management of the Orinoco crocodile in Colombia does not face genetic restrictions. In this research, we re-evaluated this proposal by using an expanded sampling and additional molecular markers (microsatellite nuclear DNA and the mitochondrial D-loop) that have successfully identified crucial information at the population level in other crocodilians. Our findings reveal, for the first time, a pattern of population structure consisting of three genetic clusters with geographical correspondence, matching three ecoregions in the Colombian Orinoco basin. This pattern might be a historical and current consequence of the ecological, bioclimatic, and/or geographical features of the ecoregion. Additionally, we examined the correspondence with the genetic clusters of the confiscated individuals that formed part of the initial population (F0) of the Colombian ex situ species population and successfully assigned most of them to one of these clusters. The genetically distinct clusters should be regarded as independent management units for conserving and managing the species in Colombia.