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Sugarcane production is constrained by several diseases, including red rot, which can adversely affect crop productivity and quality. Field trials were conducted at the Agricultural Research Station, Thiruvalla, during 2021–22, 2023–24 and 2024–25 to assess red rot resistance in sugarcane genotypes under artificial inoculation in Kerala. Eleven genotypes and three standard checks were evaluated against the virulent Colletotrichum falcatum pathotypes CF06 and CF12 using the plug and nodal cotton-swab inoculation methods. Disease reactions were recorded 60 days after inoculation. Under the plug method, Co18001, Co18002, Co18009, Co18024, CoN 18071 and CoN 18072 consistently exhibited moderately resistant reactions across the evaluated seasons and pathotypes. Co18003, Co18012, Co18013, CoVC 18061 and CoVSI 18121 showed moderately susceptible reactions. The nodal cotton-swab method produced resistant reactions in most entries, indicating lower infection pressure than the plug method. CoVC 18061 and CoVSI 18121 showed susceptibility to CF12 during the initial season under the nodal method but expressed resistance in subsequent seasons. The comparable reactions observed against CF06 and CF12 indicated consistency in genotype responses across pathotypes. The results demonstrate that disease expression is influenced by the pathotypes, season and inoculation methods, highlighting the importance of considering these factors for identifying stable resistance. The moderately resistant genotypes identified in this study warrant further validation and may be useful as genetic resources in breeding programmes aimed at improving red rot resistance in sugarcane adapted to Kerala’s agro-climatic conditions.