Objective: To explore the identity and phylogenetic relationships among the three medicinally important species of Sphaerostephanos from South India using isozymic profile. Methods: The young fronds were homogenized with 3.5 mL of ice-cold homogenizing buffer in a pre-chilled pestle and mortar. The supatant was subjected to electrophoresis as described by Anbalagan poly acrylamide gel electrophoresis. Staining solutions for isoperoxidase was prepared as per Smila method for the detection of isoenzymes. Results: A total of six different bands in five different positions with different molecular weight/Rf values and four active zones have been observed in the isoperoxidase enzyme system of Sphaerostephanos. Only one band with MW/Rf 0.399 is common to two different species i.e. Sphaerostephanos arbuscula (S. arbuscula) andSphaerostephanos unitus (S. unitus). Among the remaining four bands, two bands (Rf. 0.23, 0.47) are present in Sphaerostephanos subtruncatus (S. subtruncatus) and one distinct band has been observed individually in S. arbuscula (Rf. 0.507) and S. unitus (Rf. 0.56). Conclusions: The present preliminary molecular study through isozymic analysis shows the identity of all the three species and the present results confirm distinctness of these three species based on macro-micromorphology, phytochemistry and cytology.