The objective of this research is to evaluate the genetic variability of freshwater prawn, Macrobrachium rosenbergii. The genetic variability of freshwater prawn collected from Makassar-Sulawesi, Pangkalanbun-Kalimantan, Jambi-Sumatra, Sukabumi-Java, and GIMacro strain was examined using polymorphism of the mitochondria DNA (mtDNA) markers. Twelve composite haplotypes were detected following digestion of CO1 sequences with four endonucleases: Hae III, Rsa I, Mbo I, and Taq I. The average haplotype diversity was 0.217. Significant genetic difference was observed among freshwater prawn populations, especially among Makassar-Sulawesi population and others. Makassar Sulawesi strain has future prospect for genetic resources in breeding program.