Inter-simple sequence repeat (ISSR) analysis was used to determine the level of genetic diversity among 37 samples of Croton sublyratus Kurz from various sources, including Prachuapkhirikhan, Nakhonphanom and Prachinburi. Three RAPD primers (GGCACTGAGG, ACGACCGACG and GATGACCGCC) were used to study genetic diversity but they gave non-reproducible results. Three of ISSR primer (CA)[subscript 8]G, BSC(GA)[subscript 8] and CRN[subscript 2](CTT)[subscript 5]) gave reproducible ISSR patterns. One hundred and forty six reproducible ISSR bands were generated across the investigated species of these, 50 band (34%) were monomorphic band and 96 band (66%) were polymorphic band. Genetic variation of 37 samples of C. sublyratus were analyzed by Unweighted Pair Group Method with Arithmatic Mean (UPGMA) and the dendrogram divied C. sublyratus into 2 groups was indicated with geographical location. This study indicated that the genetic variability is higher in the species as genotypes did show distinctive leaf morphological characters under uniform conditions of growth for most of the attributes. Thus, the alteration of responses seems to be genetic variation rather than environmentally induced for C. sublyratus. In the evaluation of plaunotol content in the leaves of C. sublyratus. The analysis revealed that C. sublyratus leaves contained plaunotol in the rang 0.11-1.01% (w/w) dry weight. A specific ISSR fragment can be used for primary screening to select for high and low plaunotol content in C. sublyratus with (CA)[subscript 8]G. The result would suggest that the variation between C. sublyratus in the plaunotol content from various geographical location is governed by genetic variation.