Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Fusarium wilt, the devastating disease that ruined the 'Gros Michel' (AAA)-based banana production in the first half of the 20 th century. The occurrence of a new variant in Southeast Asia that overcomes the resistance in Cavendish clones such as 'Grand Naine' (AAA) is a major concern to current banana production worldwide. The threat posed by this new variant, called tropical race 4 (TR4), may be overcome by the introduction of resistant cultivars. However, the identification of new resistant sources or breeding for resistance is a long-term effort. Currently, the only option to control the disease is to avoid or reduce the spread of the pathogen by eradication of infected plants and isolation of infested plantations. This requires sensitive and highly specific diagnostics that enable early detection of the pathogen. A two-locus database of DNA sequences, from over 800 different isolates from multiple formae speciales of F. oxysporum, was used to develop a molecular diagnostic tool that specifically detects isolates from the vegetative compatibility group (VCG) 01213, which encompasses the Foc TR4 genotype. This diagnostic tool was able to detect all Foc TR4 isolates tested, while none of the Foc isolates from 19 VCGs other than 01213 showed any reaction. In addition, the developed diagnostic tool was able to detect Foc TR4 when using DNA samples from different tissues of 'Grand Naine' plants inoculated with TR4 isolates.