Crown gall disease, caused by the bacterium Agrobacterium tumefaciens, significantly threatens various crops, particularly fruit trees, as well as walnut (Juglans regia L.) trees. This disease leads to the formation of tumor-like growths on the trees, which can severely impact their health and productivity. The disease is particularly problematic for walnut rootstocks, such as the Paradox hybrid, which are highly susceptible to infection. In the present study, three regions of Uzbekistan (Jomboy, Bulungur, Payarik) were chosen to isolate A. tumefaciens strains on walnut and assess their virulence characteristics. Total 90 isolates were confirmed as Agrobacterium using morphological and 16S rRNA analyses; and pathogenicity tests, including the growth characteristics of the yellow colony appearance on lactose agar media poured Benedict solution, 16S rRNA sequence alignment and pathogenicity on carrot discs, Kalanchoe and Vlach rootstocks.