Experiments were undertaken to test the hypothesis that in Curcuma amada the curcuminoid content is related to ploidy level and sequence variations in the genes related to curcuminoid biosynthesis. Eight curcumin-containing accessions of C. amada and six curcumin-free accessions were analyzed. Flow cytometry revealed that the ploidy level was higher in curcumin-containing accessions than in curcumin-free accessions (average ratio 1.43). This indicates that the ploidy level correlates with the presence or absence of curcuminoids. Curcumin synthase genes (CURS1, CURS2 and CURS3) and diketide-CoA synthase genes (DCS1 and DCS2) play an important role in biosynthesis of curcuminoids. Sequencing of conserved regions of CURS1, CURS2, CURS3, DCS1, and DCS2 revealed significant sequence variations. In addition to the known CURS and DCS genes, we report 195 CURS-related sequences and 59 DCS-related sequences (including several new CURS-like and DCS-like genes) with several haplotypes for each gene. Phylogenetic analysis showed that CURS2 diverged from CURS1 and CURS3, whereas the DCS genes evolved independently of the CURS genes. DNA barcode analysis showed that the rbcL, matK and psbA-trnH intergenic region were identical in all 14 accessions and thus confirmed that all accessions belonged to C. amada.