A few number Tephrosia species have been recorded to have morphological complexity which could cause the problem to the work of taxonomist to make decisions. In order to support taxonomist a new experimental method using SDS-PAGE will be used to explore isozyme data the main purpose of this research is whether isozyme data can be used to clarify morphological differences. The present study characterizes three species of Tephrosia through its biochemical analysis including protein and polyacrylamide gel electrophoresis the increasing concentration of protein was observed in the order like Tephrosia pentaphylla, Tephrosia villosa and Tephrosia purpurea. Two isozyme systems have been recorded from the Tephrosia species alkaline phosphatase and esterases along with that molecular Weight determination was carried out as these species are economically important and to identify the difference between them.