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Fusarium dieback is a prevalent disease of tea (Camellia sinensis (L.) Kuntze) in India and China. The pathogen affects plucking shoots and causes complete crop loss under favourable conditions. Although, other fungal diseases that infect tea were widely explored for pathogen identification and characterization, little is known about Fusarium dieback in major tea growing regions of India. Therefore, in this study 24 isolates of Fusarium species causing dieback on tea in 24 tea gardens of Assam and North Bengal were isolated and characterized through micro-morphological and molecular analysis using multi-locus DNA sequences to determine whether multiple species of Fusarium are responsible for dieback disease. Molecular analysis revealed the association of four species of Fusarium , namely F. concentricum, F. solani, F. fujikuroi, and F. oxysporum. The most dominant species was F. concentricum (62.5%), whereas the occurrence frequencies of F. solani, F. fujikuroi, and F. oxysporum were 20.8%, 12.5%, and 0.04%, respectively. All Fusarium isolates were pathogenic to tea leaves when individually and co-inoculated, with F. concentricum HPE0F10 and NHT0F17 exhibiting 19.3 mm lesion length being the most virulent isolates. The growth rate of the pathogen was positively correlated with lesion lengths (P < 0.001). Co-infection of two or multiple Fusarium species enhanced the disease severity and quicker disease development compared to single inoculation. All the isolates showed diverse genetic backgrounds based on Inter Simple Sequence Repeat (ISSR) primers and principal coordinate analysis. In fungicide screening test, all isolates except HPE0F10, NDS0F18, and SNY0F22 were insensitive to M1 fungicide (copper oxychloride). Isolates GNP0F8 and LKW0F16 were insensitive to MBC + M3 (Carbendazim + Mancozeb), whereas HPE0F10, NLG0F11, IND0F13, and NLW0F19 were insensitive to DMI (respective each two to Hexaconazole and Valextra) fungicides. Notably, all the isolates were susceptible to propiconazole. Further, ISSR segregated isolates insensitive to MBC + M3 and M1 fungicides into two separate clusters, representing their distinctiveness. The fungicide screening results indicate that fungicide resistance can be managed by rotating different modes of action of fungicides against multiple Fusarium species causing tea dieback. • Fusarium concentricum, F. fujikuroi, F. oxysporum, and F. solani were identified as causal agents of tea dieback. • The identification was confirmed by multi-locus phylogenetic analysis. • Fusarium isolates showed variable pathogenicity through individual and co-inoculation tests. • ISSR primers revealed diverse genetic backgrounds among 24 Fusarium isolates. • Propiconazole was effective against all Fusarium isolates tested. [ABSTRACT FROM AUTHOR]