While walnuts are grown worldwide, two species are predominantly cultivated in China, Juglans regia and Juglans sigillata. J. regia is a widespread species, while J. sigillata is an indigenous species mainly distributed in southwestern China. In Tibet, unique plateau climatic conditions and relatively low-intensity agriculture aid walnuts to preserve a great variety of genetic resources. Knowing the genetic diversity and genetic structure of walnut populations in Tibet is essential when planning genetic conservation and screening for superior germplasm resources. The objectives of this study were to reveal the genetic diversity and genetic structure of walnut populations in Tibet and to infer the relationship between two species, J. regia and J. sigillata, using 12 molecular markers. The results based on screening 209 walnut trees from nine populations showed that the level of polymorphism is moderately high. However, the number of rare alleles (allele frequency <0.05) was higher than that reported in previous studies. An analysis of molecular variance revealed that significant genetic variation existed both among populations of J. regia (10.25 %, p < 0.0001) and J. sigillata (11.07 %, p < 0.0001) and between the two species (7.91 %, p < 0.0001). A Bayesian approach divided the 209 walnut samples into two clusters and presented the differentiation pattern of these two species. The assignment analysis revealed the presence of J. regia × J. sigillata hybrids among the sampled individuals. The results suggest unique germplasm preservation among the walnut populations in Tibet and that introgression between J. regia and J. sigillata may account for the convoluted boundary between the two species.