Moko disease in banana is a bacterial wilt caused by strains within Ralstonia solanacearum sensu stricto. The disease is endemic to Central and South America but has spread to the Philippines and peninsular Malaysia. Detecting new incursions early in Moko-free banana production regions is of utmost importance for containment and eradication, as Moko management significantly increases costs in banana production. Molecular studies have supported the classification of R. solanacearum sensu stricto into phylotypes IIA, IIB, and IIC, each comprising various sequevars based on nucleotide divergence of a partial sequence within the endoglucanase gene. Moko disease in banana is caused by strains classified as sequevars 6, 24, 41, and 53 within phylotype IIA and sequevars 3, 4, and 25 within phylotype IIB. To ensure accurate diagnostic assays are available to detect all Moko sequevars, we systematically validated previously published assays for Moko diagnostics. To be able to identify all sequevars, including the latest described sequevars, namely IIB-25, IIA-41, and IIA-53, we developed and validated two novel assays using genome-wide association studies on over 100 genomes of R. solanacearum sensu stricto. Validations using 196 bacterial isolates confirmed that a previous multiplex PCR-based assay targeting sequevars IIB-3, IIB-4, IIA-6, and IIA-24 and our two novel assays targeting sequevars IIB-25, IIA-41, and IIA-53 were specific, reproducible, and accurate for Moko diagnostics.