Boesenbergia rotunda or locally known as 'Temu kunci' is renowned to possess some important bioactive compounds that are promising for pharmacological applications. It is essential to obtain structural information of the inner parts of the in vitro roots since growing these roots undertake various anatomical and morphological changes that influence their cells activities and nutrient uptake processes. Although root functions are thought to be significant for the growth of shoot, the morphological and anatomical knowledge of adventitious root for this plant are limited. This particular study aims to investigate and compare anatomical structures of in vitro adventitious root derived from in vitro plantlet with root derived from shoot bud of B. rotunda. Histological sections using resin were done to study the anatomical of adventitious root of B. rotunda. The root samples were fixed in a Glutaraldehyde-Paraformaldehyde-Caffeine (GPC) fixative, dehydrated, infiltrated, embedded, cut, stained and mounted with Surgipath mounting medium for observations under light microscope. From the histological observations, adventitious roots of B. rotunda had shown the presence of all the three main tissue systems and had the same internal structures containing epidermis, exodermis, suberized sclerenchyma cells, cortex and stele. Both adventitious roots derived from in vitro plantlet and shoot bud showed normal growth morphologically, had anatomically same normal cell structures and arrangements.