We carried out the morphological, biochemical and molecular characterization of 20 indigenous Joha (aromatic) rice cultivars of Assam. Distinctiveness, Uniformity and Stability (DUS) characterization of the cultivars revealed polymorphism in thirty-seven traits, establishing distinctiveness for their utilization in breeding programmes. Unweighted Neighbour Joining (UNJ) clustering based on usual Euclidean distances for the polymorphic markers grouped the cultivars into three multi-genotypic clusters. The Joha rice cultivars showed highly significant differences for all the quantitative traits except for panicle length. The genotypic and phenotypic coefficients of variability (GCV & PCV) were high for grain yield ha− 1 (24.62 & 24.85%) and filled grains panicle− 1 (23.69 & 25.02%). All the traits except days to flowering and maturity, flag leaf breadth and spikelet fertility exhibited high heritability along with high to moderate genetic advance, indicating the predominant role of additive gene action. Mahalanobis D2 analysis revealed three multi-genotypic and four mono-genotypic clusters of the cultivars. The cultivars' average polyunsaturated fatty acids were 37.9% oleic acid, 39.22% linoleic acid and 0.5% linolenic acid. The fatty acid profile of Local Joha was superior to the other cultivars as it showed a high level of linoleic and linolenic acid and low saturated fatty acid content. Kon Joha 4 and Ronga Joha contained the highest iron (82.88 mg kg− 1) and zinc (47.39 mg kg− 1), respectively, while protein content of Kon Joha-1 and amylose content of Harinarayan were the highest. Joha-Bihpuria showed the highest gel consistency of 140.50 mm. Kalijeera, Kunkuni Joha, Kon Joha-5, Manimuni Joha and Kon Joha-2 accorded a strong aroma. PCR amplified 174 alleles with a mean value of 2.64 across the 66 polymorphic SSR markers. PIC values ranged from 0.091 to 0.698, with an average of 0.326. The highly informative (PIC > 0.50) markers were RM316, RM283, RM585, RM1388, RM3562, RM171, R1M30, RM118, RM11and RM29 for identification of the twenty aromatic rice cultivars. The UNJ clustering based on Jaccard's coefficients classified the 20 cultivars into three distinct clusters with eight, ten and two entries.