We assessed the spatial distribution of the genetic variability of Melanosuchus niger from 11 localities in South America using 1,027 base pairs of the mitochondrial cytochrome b gene. Screening 132 animals, we found 41 haplotypes, high values of genetic diversity, low values of nucleotide diversity and significant deviations from neutral expectation of allelic frequencies in some localities. Mantel test and nested-clade analysis indicated that isolation-by-distance was an important population dynamic for the species as a whole. Wright's fixation indexes analyses showed that hydrogeographically separated populations from French Guiana together with Amapá state population in Brazil are genetically differentiated from all other populations that are found in the Amazon drainage basin. These indexes also disclosed that the population from Ecuador is genetically differentiated in relation to the populations from Brazil, Peru and French Guiana. Within the Amazon Basin little differentiation exists, and genetic and geographic distances are not correlated. Demographic data as well as population genetic data suggest that M. niger is recovering in some protected regions. However, part of this apparent recovery may be owing to the movement of animals into protected regions. J. Exp. Zool. 309A:600–613, 2008. © 2008 Wiley-Liss, Inc.