Vanilla (Vanilla planifolia), a tropical orchid cultivated for its pleasant flavour, is a native of Mexico, and an introduced crop to other countries. Continuous vegetative propagation and lack of sufficient variations in the gene pool hampers crop improvement programmes. Introduction of somaclonal variation through callus cultures has been attempted to broaden the narrow genetic base. A callus induction and in vitro plant regeneration system has been optimized from both vegetative and reproductive tissues. The best results were obtained using vegetative tissues and over 80% callusing was achieved in Murashige and Skoog’s medium supplemented with 4.44μM BA and 2.68μM NAA. Callus differentiated into shoots which could be multiplied successfully in 1: 12 ratio in a combination of 4.44μM BA and 2.46μM IBA, when supplemented to MS medium. In vitro rooting was induced with an efficiency of 100% in basal MS media devoid of any growth regulators. This ability of dedifferentiated tissue to regenerate is a crucial prerequisite for future genetic transformation experiments. The protocol was successfully extended to the endangered wild species, V. aphylla, offering the potential of applying the protocol for mass multiplication as well as induction of variations in Vanilla species, in a limited time. Preliminary studies on the callus regenerants indicated variations in morphology and RAPD profiles.