Abstract: Juglans regia L. and J.sigillata Dode. are two mainlycultivated walnut species in China, and the later is a unique species distributed in the southwest China. Furthermore, the argument about relationship of the two species remains undetermined. Five populations of J.regia and J.sigillata were chosen to investigate their relationship with ITS and SSR markers. Result of ITS sequence only detected 1 nucleotide difference between the two species. Based on 65 alleles generated with SSR, genetic distance between populations was calculated. The closest genetic distance was 0.119 found between SJC and QZ population, and the furthest distance was 0.552 found between MN and HS population. The genetic distance between intraspecies was similar to that between interspecies, and the difference between two species was quite small and was not significant. Those results suggested that the two species might be different ecotypes, which provided a scientific reference for revealing the evolutional and genetic relationship and genetic characteristics of walnut plants.