A major constraint on castor crop productivity is wilt disease caused by Fusarium oxysporum f. sp. ricini (For). In this study, morphological, pathogenic and molecular features of F. oxysporum f. sp. ricini isolates collected from different castor-growing agro-climatic zones of India were analyzed. Eighty-nine F. oxysporum f. sp. ricini isolates were obtained from wilt-infected root samples of castor plants from different states of India viz., Andhra Pradesh, Bihar, Gujarat, Haryana, Karnataka, Madhya Pradesh, Maharashtra, Odisha, Rajasthan, Telangana and Tamil Nadu. Morphology assessment in terms of variability in radial growth, size of micro and macroconidia and number of septa, grouped the isolates into 5 clusters in biplot analysis. A genotype-by-isolates bi-plot analysis ranked genotypes for pathogen susceptibility, with JI-35 being closest to the ideal and most susceptible, and DCS-9, and Kranthi being less susceptible. In contrast, 48–1 and JI-258 showed resistance, while Haritha and VP-1 exhibited intermediate susceptibility levels. K-means cluster analysis found 5 groups for pathogenic variability studies of isolates. The six isolates viz., For-12-5 (61.8 %), For-12-10 (50.5 %), For-13-47 (51.4 %), For-113 (86.1 %), For-13-33 (77.9 %) and For-53 (62.2 %) recorded as highly virulent isolates against wilt disease which showed >50 % average wilt incidence mean value across cultivars. Out of 40 ISSR primers tested, 11 primers gave polymorphic bands and analysis of genetic variability among 36 F. oxysporum f. sp. ricini isolates showed polymorphism with PIC, heterogygosity index, effective multiplex ratio, Marker index, and Shannon Diversity Index which ranged (in %) between 0.22 and 0.69, 0.19–0.44, 11.09–27.9, 6.14–8.98, and 0.78–3.04 respectively. The dendrogram analysis revealed 53 % similarity among the isolates studied and these isolates grouped broadly into 6 clusters. Variability among isolates was significant in morphological, pathogenic and molecular characters of F. oxysporum f. sp. ricini.