Antheraea mylitta Drury, the semi-wild silk-producing lepidopteran insect commonly known as tasar silkworm is unique to India and is distributed over a wide tropical forest range covering the states of Andhra Pradesh, Bihar, Chhattisgarh, Madhya Pradesh, Maharashtra, Orissa, and Uttaranchal. The populations found in different areas are know by their specific local names and are considered as different ecotypes, but it is difficult to separate the populations on the basis of morphological and life-cycle traits and thus molecular characterization was attempted. The present communication relates to the results obtained from the analysis of polymorphism unraveled by twelve ISSR primers for 11 populations of A. mylitta belonging to six ecotypes and 41 individuals of Railey ecotype collected from five zones of Dandakarnya forest in Madhya Pradesh. This communication, further, presents molecular evidences on genetic differences between eleven ecotype populations and highlights the genotypic diversification of a single ecotype into further separate discrete gene pools. The canonical discriminant function analysis revealed grouping of the five populations of Railey ecotype into two "clumps," while accessions of other ecotypes stood separated from each other. The Railey populations on detailed study, further, revealed separation of two (Tokapal and Nangur) populations into discrete gene pools and the other three (Kondagaon, Darba, and Tongpal) populations, overlapped in spite of larger geographic distance between them. The analysis also identified nine markers, which can be utilized to characterize specific population and will be of help to follow the ongoing genetic changes triggered by various ecological factors and human influences on the Railey ecotype.