We evaluated the selected populations of eighteen open-pollinated short-day onion genotypes for FBR-Fusarium basal rot (Fusarium oxysporum f. sp. cepae-FOC) susceptibility; population genetic variation via ISSR marker; and transcriptome analysis using qRT-PCR with six novel selected marker genes: R1, R5, RGA29, lectin, LOX, and Osmotin, at tree time post inoculation (wpi). Screening for resistance showed the average severity between 4.7 and 88.9%; of which, the lowest one was in ‘Saba’ and ‘Saba – HS (6.7 and 4.7%, respectively). ISSR analysis recorded 226 amplified bands, of which 160 bands showed polymorphism, of which ISSR1 and ISSR10 primers showed the best performance. We also found that following inoculation with FOC could regulate defense-related marker genes; R1, PR5, Lectin, LOX, Osmotin, and RGA29 in resistant onion “Saba” and ‘Saba’-HS in comparison to susceptible and controls, non-inoculated ones ranging from 1.23 to 6.99 -fold significantly. Surprisingly, marker genes; Lectin, LOX, and Osmotin were also expressed to FOC simultaneous, though basically are resistance to other biotic and abiotic stress: Lectin to Rhizoctonia solani, Aphid, and major sap-sucking pests; LOX to root-knot and cyst nematode, Heterodera glycines; Osmotin to drought stredd, and oxidative burst in plants. This indicates the double, and or multiple roles of our selected marker genes covering two or more functions at a time. The findings introduce newly resistant onion genotypes, and also can be used in management programs to reduce damages caused by FOC disease. Cumulatively, the transcriptome-data provide novel-insights into the response of onions for improving onion-breeding to FOC.