One of the main hypotheses to explain the origin of Amazonian diversity is the barrier effect of the rivers known as the riverine-barrier hypothesis, which suggests that riverine barriers isolated once continuous populations leading to differentiation and speciation. In this context, we studied the genetic structure of Pristimantis latro, a newly described species that inhabits a region under marked anthropic pressure due to expansive livestock, illegal mining, and hydroelectric dam construction. The DNA was extracted from 52 P. latro individuals and then amplified via polymerase chain reaction (PCR) using the mitochondrial 16S rRNA and COI markers. To infer the time of divergence between the P. latro localities, we built a species tree and performed an analysis of molecular variance (AMOVA) to infer the genetic differentiation between and within the P. latro populations. We found that P. latro has a marked genetic structure in the populations of the right and left margins of the Xingu River and within the Tapajós-Xingu and Xingu-Tocantins interfluvial regions and that the time of divergence between the populations of the East and West banks of the Xingu River occurred approximately 380,000 years ago. This pattern of genetic structure corresponds to that reported in recent articles for the Pristimantis genus evidencing that species without tadpoles exhibit a genetic structure explained by the hypothesis of rivers as barriers.