In June 2019, root mat disease was observed in hydroponically cultivated tomatoes in Jinju, South Korea, which occurred in at least 30% of the plants in the greenhouse. To isolate the causal bacteria, 10 g of infested tomato root mat sample was ground with 50 mL of sterile water. A 100-µL aliquot of the homogenate was serially diluted and spread on Mannitol-Glutamate (MG) medium amended with 0.1% yeast extract (MGY) and incubated at 28°C for 48 hours. Fifteen dominant colonies that formed on the MGY medium were purified and subjected to diagnostic polymerase chain reaction (PCR) based on the virD2-ipt gene loci. Because Ti-plasmid harbors both virD2 and ipt genes, Ri-plasmid-borne Agrobacterium species with only virD2 can be differentiated using virD2-ipt PCR. To amplify virD2, the primers 5'-ATG CCC GAT CGA GCT CAA GT-3' and 5'-TCG TCT GGC TGA CTT TCG TCA TAA-3' were used; for ipt amplification, the primers 5'-GAT CG(G/C) GTC CAA TG(C/T) TGT-3' and 5'-GAT ATC CAT CGA TC(T/C) CTT-3' were used. Amplification involved an initial 94°C for 1 min and then 40 cycles at 94°C, 50°C, and 72°C for 1 min at each temperature, with a final 5-min extension at 72°C. For three strains (GNIY2, J10, and J11), virD2 PCR products, but no ipt PCR products, were identified, indicating that three strains are Ri-plasmid-borne Agrobacterium species. A pathogenicity test was performed on 2-week-old tomato plants. Bacteria isolates (GNIY2, J10, and J11) cultured overnight in LB were made into a bacterial suspension (107 cfu/mL) in 50 mM phosphate buffer. Five tomato seedling roots were cut with sterilized scissors and soaked in each bacterial suspension for 1 hour. Phosphate buffer was used as a negative control. The inoculated tomato seedlings were transplanted in new pots and placed in a greenhouse with 25°C day and 20°C night temperature set points in natural light. After 9 weeks, all inoculated tomato plants produced overgrown roots, while the negative control plants had no symptoms. To satisfy Koch's postulates, re-isolation was performed from the diseased tomato and was the re-isolated bacteria were subject to partial 16S rDNA sequencing. Biovar tests performed as previously described revealed that all three isolates were biovar 1. A representative strain (GNIY2) was deposited in the Korean Agricultural Culture Collection (KACC 21759). To confirm the identity, four housekeeping genes of KACC 21759 were sequenced (16S rRNA, trpE, rpoB, and recA) and deposited in GenBank (accession nos. MT071560, MT444428, MT444429, and MT444430). Multilocus sequence analysis performed as previously described showed that the KACC 21759 strain was grouped in Agrobacterium genomospecies 4. This is the first report on mat root disease caused by Agrobacterium biovar 1 in hydroponic tomatoes in South Korea.