Jatropha curcas L. is a potential bioenergy crop but has a lack of improved cultivars with high yields and oil content. Therefore, increasing our understanding of J. curcas germplasm is important for designing breeding strategies. This study was performed to investigate the genetic diversity and population structure of Indonesian J. curcas populations from six different islands. To construct a reference, we de novo assembled the scaffolds (N50 = 355.5 kb) using 182 Gb Illumina HiSeq sequencing data from Thai J. curcas variety Chai Nat. Genetic diversity analysis among 52 Indonesian J. curcas accessions was conducted based on yield traits and single nucleotide polymorphism (SNP) markers detected by mapping genotyping-by-sequencing reads from Indonesian population to Chai Nat scaffolds. Strong variation in yield traits was detected among accessions. Using J. integerrima as an outgroup, 13,916 SNPs were detected. Among J. curcas accessions, including accessions from other countries (Thailand, the Philippines and China), 856 SNPs were detected, but only 297 SNPs were detected among Indonesian J. curcas populations, representing low genetic diversity. Through phylogenetic and structural analysis, the populations were clustered into two major groups. Group one consists of populations from Bangka and Sulawesi in the northern part of Indonesia, which are located at a distance of 1572.59 km. Group two contains populations from islands in the southern part: Java, Lombok-Sumbawa, Flores and Timor. These results indicate that introduction of diverse J. curcas germplasms is necessary for the improvement of the genetic variation in the Indonesian collections.