Populations of large and medium mammals in tropical rainforests have been subdivided and reduced in numbers due to habitat loss and fragmentation. Bottlenecks and restrictions to gene flow may lead to structured populations with low genetic variation, negatively affecting long-term population persistence. Here, we analysed the genetic variability and population structure of a keystone ungulate species, the white-lipped peccary (Tayassu pecari). This species is considered “vulnerable” in the IUCN Red List because of local extinctions and population declines throughout its distribution. We collected a total of 380 samples and genotyped 15 microsatellites from areas with different degrees of fragmentation from Pantanal, Cerrado, and Atlantic Forest in Brazil. We expected to find highly structured populations with low genetic diversity in Cerrado and Atlantic Forest because these biomes are more threatened than Pantanal. Contrary to our expectations, we found populations with weak or no signal of genetic differentiation and similar levels of genetic diversity in the three biomes. In addition, we detected no evidence of inbreeding and recent bottlenecks in all populations analysed. Recent isolation or persistence of gene flow among the sampled populations may explain our results. This study serve as a starting point for the genetic monitoring of these populations, and although no population bottleneck, inbreeding and loss of genetic diversity have been detected, the increasing threats to the biomes studied here should be considered in the species conservation and management plans.