Abstract Nutmeg (Myristica fragrans Houtt; family: Myristicaceae) is an important tree spice of great export value due to the presence of secondary metabolites with scope in health, wellness and cosmetic industries. Its dioecious in nature due to which it is propagated by budding and grafting which requires specialised skill and as a result authentic planting material production is very expensive. Based on the constant demand from farmers and stakeholders for a suitable, fast and easy technique for determining the authenticity of nutmeg varieties, an attempt was made to develop morphological and molecular markers for the identification of three commercial varieties that are in high demand in India; viz. IISR Vishwashree, IISR Keralashree, Sindhushree and a monoecious nutmeg (MN) accession, along with their biochemical profiles. Among the various morphological characters as per distinctiveness, uniformity and stability guidelines, the varieties showed distinctness mainly in leaf shape, shape of female flowers, fruit shape and seed shape. Out of 35 ISSR primers screened, six primers viz., IS 02, ISSR 12, ISSR 05, ISSR 14, ISSR 01 and UBC 834 generated clear, unique reproducible polymorphic bands capable of distinguishing the varieties. Among the varieties analysed, MN was found to be superior in terms of seed butter and essential oil in nut, mace and leaves. IISR Vishwashree was on par with MN in nut essential oil content. IISR Keralashree was superior in terms of oleoresin in nuts and mace. The morphological and molecular markers identified may be used for effective checking of authenticity of planting materials of commercially grown nutmeg varieties.