A study of genetic diversity in Pongamia pinnata representing 33 candidate plus trees (CPT) was undertaken in five agro-ecological zones of Southern Peninsular India. Eleven primer combinations generated 532 bands with an average of 48 bands per primer combination. E-ACG/M-GCC showed the highest polymorphism of 98.80 %. E-ACG/M-GCC, E-CAA/M-CTG, and E-CAG/M-GTG with higher polymorphic information, marker index, and resolving power were found to be effective in detecting unique bands. Nei's gene diversity and Shannon's indices of 0.159 and 0.247, respectively, indicated the presence of high gene diversity in southern dry and transition zones of Karnataka. The inter-population differentiation between zones (GST) was 0.296. The level of gene flow (Nm) estimated was 1.34. Analysis of molecular variance indicated that 44.78 % of variance occurred within the provenance when compared to variations among agro-ecological zones (22.22 %) and among provenances within zones (33 %). Based on un-weighted pair group method using arithmetic averages method, the pongamia accessions were grouped into three major clusters and four sub-groups depending on their geographical locations. The diverse CPT identified in this study could be selected and used for tree breeding, germplasm conservation, and biofuel program in India and other tropical countries.