Cassia auriculata is considered to be one of the important dye yielding and medicinal plants in India. In the present study seeds from fourteen different localities were collected all over India and nine enzymes were screened by native polyacrylamide gel electrophoresis (PAGE) technique and thirty-four putative loci were totally detected. Cluster and factor analyses indicated that there are two major distinct groups or clusters, and thus, seeds collected from a few different localities are enough to capture the genetic variation held by this species. Also isozyme analysis is a reliable, efficient and effective marker technology for determining genetic variations in C. auriculata. © 2005 Academic Journals.