The behavior of naturally virulent Meloidogyne isolates toward the tomato resistance gene Mi in major tomato-growing areas in Israel was studied for the first time. Virulence of seven selected isolates was confirmed over three successive generations on resistant (Mi-carrying) and susceptible (non-Mi-carrying) tomato cultivars. Diagnostic markers verified the predominance of Meloidogyne javanica among virulent isolates selected on resistant tomato cultivars or rootstocks. To better understand the determinants of nematode selection on Mi-carrying plants, reproduction of Mi-avirulent and virulent isolates Mjav1 and Mjv2, respectively, measured as eggs per gram of root, on non-Mi-carrying, heterozygous (Mi/mi) and homozygous (Mi/Mi) genotypes was evaluated. Although no reproduction of Mjav1 was observed on Mi/Mi genotypes, some reproduction was consistently observed on Mi/mi plants; reproduction of Mjv2 on the homozygous and heterozygous genotypes was similar to that on susceptible cultivars, suggesting a limited quantitative effect of the Mi gene. Histological examination of giant cells induced by Mi-virulent versus avirulent isolates confirmed the high virulence of Mjv2 on Mi/mi and Mi/Mi genotypes, allowing the formation of well-developed giant-cell systems despite the Mi gene. Analysis of the plant defense response in tomato Mi/Mi, Mi/mi, and mi/mi genotypes to both avirulent and virulent isolates was investigated by quantitative real-time polymerase chain reaction. Although the jasmonate (JA)-signaling pathway was clearly upregulated by avirulent and virulent isolates on the susceptible (not carrying Mi) and heterozygous (Mi/mi) plants, no change in signaling was observed in the homozygous (Mi/Mi) resistant line following incompatible interaction with the avirulent isolate. Thus, similar to infection promoted by the avirulent isolate on the susceptible genotype, the Mi-virulent isolate induced the JA-dependent pathway, which might promote tomato susceptibility during the compatible interaction with the homozygous (Mi/Mi) resistant line. These results have important consequences for the management of Mi resistance genes for ensuring sustainable tomato farming.