Corynespora cassiicola isolates are periodically sourced from infected stands in rubber plantations throughout Malaysia. The severity of C. cassiicola infection varies among the clonally propagated Hevea brasiliensis. In this study, 26 C. cassiicola isolates collected from 1988 until 2006 were analysed for the gene encoding cassiicolin an important fungal effector involved in the Corynespora leaf fall disease. Cassiicolin gene was successfully amplified by polymerase chain reaction (PCR) from 13 C. cassiicola isolates. Deduced protein sequences revealed that 12 isolates harbour the Cas5 gene while one isolate (CKT05D) contains the Cas4 gene. The other 13 isolates in which no Cas gene was detected were classified as Cas0. PCR amplifications were also performed on all 26 isolates using primers specific to the ITS1-5.8S-ITS2 region of ribosomal DNA, the random hyper variable loci ga4 and caa5, and the actin locus act]. A phylogenetic analysis performed on the 26 isolates using four loci (rDNA ITS, caa5, ga4 and act]) revealed three clusters. Cluster 1 encompasses all Cas5 isolates plus two Cas0 isolates, CSDI and CBPP2. Cluster 2 is represented by the single Cas4 isolate (CKT05D) and clusters 3 groups all the other as isolates. When placed in a previously described phylogenetic tree of C. cassiicola isolates from various geographical origins and hosts, clusters I and 3 fell in clades B4 and A4 respectively. However, CKT05D (Cluster 2) was not placed in a highly supported clade. A detached Hevea leaf assay was performed on six clones with a selection of four C. cassiicola isolates originating from the different clusters that showed varying degree of infectivity. Interestingly, severe necrotic lesion was discerned in most of the clones inoculated with CKT05D (toxin class Cas4) while the other isolates caused moderate to mild infection on all the tested clones.