Knowledge on the genetics of maydis leaf blight (MLB) is crucial to breed the resistant maize cultivars to combat disease epidemics as a sustainable and cost-effective approach. The present investigation was framed to understand the genetics of MLB resistance in subtropical maize. Two contrasting genotypes CM119 (susceptible) and SC-7-2-1-2-6-1 (resistant) were used to generate six genetic populations, namely P₁, P₂, F₁, F₂, BC₁P₁ and BC₁P₂, and evaluated in three target environments for MLB resistance under artificial epiphytotic condition. The CM119 and SC-7-2-1-2-6-1 showed susceptible and resistant reactions with mean disease reaction of 3.89–3.98 and 1.88–2.00, respectively. The derived generations, namely F₁, F₂, BC₁P₁ and BC₁P₂ showed mean disease reaction of 2.15–2.28, 2.44–2.51, 2.19–2.24 and 2.22–2.28, respectively in the test locations. The segregating generations (F₂: 0.35–0.37; BC₁P₁: 0.24–0.29 and BC₁P₂: 0.17–0.20) showed variation for MLB disease resistance over the parental and first filial generations (P₁: 0.11–0.17; P₂: 0.08–0.13 and F₁: 0.12–0.14). The genetic analysis of MLB resistance revealed the nonallelic interactions of duplicate epistasis type across the test locations. Among the gene interactions, dominance × dominance [l] effect was predominant over additive × additive [i] and additive × dominance [j] effects. The segregation analysis and the prediction of the number of major loci revealed at least two major genes associated with MLB tolerance in subtropical maize. Our investigation paved the foundation for the improvement of subtropical maize germplasm of MLB resistance.