Background <p>Recently identified as an emerging pathogen, tomato mottle mosaic virus (ToMMV), belonging to the <i>Tobamovirus</i> genus, has become a major concern for global tomato cultivation. To better understand its genetic characteristics and support future research, we investigated a newly collected ToMMV isolate from symptomatic tomato plants in China and aimed to develop a reliable infectious clone for downstream studies.</p> Methods and Results <p>A ToMMV-positive tomato sample from China was sequenced, revealing 98.5% nucleotide identity with a previously reported isolate from Shandong Province, China. A full-length cDNA infectious clone of this isolate was constructed using Gibson Assembly. Agroinfiltration of the clone into <i>Nicotiana benthamiana</i> and tomato (<i>Solanum lycopersicum</i>) plants resulted in the successful induction of typical disease symptoms in both host species. Pathogenicity assays using sap inoculation confirmed that the clone retained virulence comparable to field isolates. A preliminary evaluation of several tomato cultivars commercially cultivated in China demonstrated varying levels of susceptibility to the virus.</p> Conclusions <p>This study demonstrates that Gibson Assembly provides an efficient and reliable approach for constructing an infectious clone of ToMMV. The resulting clone reproduced disease symptoms and pathogenicity, providing a valuable tool for future research on ToMMV biology. The differential cultivar responses identified in the screening further highlight potential sources of resistance and will aid future efforts in pathogenicity studies, host–virus interaction analyses, and tomato resistance breeding.</p>