ABSTRACT:Methods have been devised for detecting polymorphisms in the D-loop regions using the PCR followed by restriction enzyme digestion to reveal restriction fragment length polymorphism. Total DNA were extracted from the white blood cell of swamp buffaloes. Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of local swamp buffalo from Lamphayaklang Livestock Research and Breeding Station in Thailand were analyzed using three enzymes (Ava lI, Bam H I and Hae III) which recognize 4 and 6 nucleotides. Among the 49 animals were analyzed, Ava II, BamH I and Hae III revealed different polymorphisms. Ava II showed two types of cleavage pattern. BamH I showed two type of cleavage pattern. And Hae III showed four types of cleavage pattern. The polymorphic fragments patterns for these three restriction endonuclease (Ava II-BamH I-Hae III) revealed five types of D-loop mtDNA in all the animals examined. These preliminary results indicated that polymorphism in the D-loop region of swamp buffalo mtDNA can be revealed by PCR and restriction enzyme analysis. And these enzymes would be able to show the genetic difference among the swamp buffaloes. These preliminary results indicated that polymorphism in the D-loop region of swamp buffalo mtDNA can be revealed by PCR and restriction enzyme analysis. And these enzymes would be able to show the genetic difference among the swamp buffaloes.