Tomato yellow leaf curl virus (TYLCV) infections result in decreased tomato growth and reduced yields, and the production is almost entirely lost if plants are infected during early growth. ‘Kyo-temari’ is the commercial name for the parthenocarpic tomato ‘MPK-1’, which has been vegetatively propagated and distributed to local farmers in Kyoto City for commercial cultivation. During the winter of 2013, the typical yellow leaf curl symptoms of TYLCV were observed in 10 parthenocarpic tomato cultivars, including ‘MPK-1’, maintained as mother stock for vegetative propagation at Kyoto University. When microtissue direct polymerase chain reaction was conducted, a begomovirus-specific amplicon was detected in the plants with yellow leaf curl symptoms. Sequencing and phylogenetic analysis clarified that a TYLCV-Mild isolate was infecting the parthenocarpic tomatoes. Because signals of TYLCV were not detected in the shoot apical meristems (SAMs) of TYLCV-infected ‘MPK-1’ by in situ hybridization, elimination of TYLCV was conducted by regenerating plants from leaf primordia (LP)-free SAMs of parthenocarpic tomato cultivars. By combining the LP-free SAM culture and in vitro grafting, TYLCV-free plants were obtained in approximately three months. The technique developed in this study will contribute to the efficient elimination of TYLCV from vegetatively propagated parthenocarpic tomatoes.