The black walnut Juglans neotropica is a forest species characterized by being a monoecious and deciduous tree with a long life. This species has great ecological, environmental, and economic value, playing a fundamental role in the ecosystem. According to the IUCN, J. neotropica is threatened by anthropogenic activities that have drastically affected its distribution. In this study, the plastid intergenic spacer marker trnS-trnfM was amplified from 74 J. neotropica samples from eight locations in Amazonas region (Peru) to determine its haplotype network, genetic diversity, and genetic divergence. The results revealed that J. neotropica from Amazonas region showed i) a lineage composed of the eight populations embedded into the Rhysocaryon section; ii) three genetic groups within the haplotype network with the presence of an ancestral haplotype (H1) and possibly candidates for new taxa; iii) a high divergence between the populations of Molinopampa and Luya (1.62–2.64% of p-distance); iv) populations with high genetic diversity indices (Levanto = 0.32, Molinopampa = 0.41) with constant threats from anthropogenic activities; and v) high genetic structuring within populations (Fst = 0.04). Overall, these results collectively support a scenario of high variability with limited interpopulation genetic exchange. Our findings provide previously unavailable insights into the vulnerability of the black walnut J. neotropica by (i) quantifying the genetic consequences of human-induced habitat fragmentation and (ii) establishing baseline diversity metrics for future monitoring. These results directly inform in situ conservation priorities by identifying populations harbouring unique alleles that warrant immediate protection. Finally, further research should include nuclear markers (e.g., microsatellites or RAD-seq) to support our findings.