The edible tubers from different species of Dioscorea are a major source of food and nutrition for millions of people. Some of the species are medicinally important but others are toxic. The genus consists of about 630 species of almost wholly dioecious plants, many of them poorly characterized. The taxonomy of Dioscorea is confusing and identification of the species is generally problematic. There are no adequate anatomical studies available for most of the species. This study is aimed to fill this gap and provides a detailed investigation of the anatomy and micro-morphology of the rhizomes and tubers of five different species of Dioscorea, namely D. balcanica, D. bulbifera, D. polystachya, D. rotundata and D. villosa. The primary features that can help in distinguishing the species include the nature of periderm, presence or absence of pericyclic sclereids, lignification in the phloem, types of calcium oxalate crystals and features of starch grains. The descriptions are supported with images of bright-field and scanning electron microscopy for better understanding of these species. The diagnostic key of anatomical features included in this paper can help distinguish the investigated species unambiguously. Additionally, HPTLC analyses of authentic and commercial samples of the five species are described.