Abstract : Analyzing the variations of nucleotide sequences in D-loop regions mtDNA can determine the identity of specific individuals or populations and maternal kinship. The Madurese population is an ethnic group that maintains its traditional customs in harmony with the religions they adhere to and endogamous marriage as their marriage pattern. This study applied PCR amplification and sequencing strategies on HVS-1 143 bp (nt 16268-16410) and HVS 2 126 bp (nt 34-159) of mtDNA D-loop regions. This study used buccal swab samples collected from 50 pure Madurese families consisting of a mother and two children. The homology analyses of female-female, male-female, and male-male siblings showed 11 variants or morphs in 126bp HVS-2 D-Loop mtDNA (nt 34-159). The highest variant were: female-female siblings (129GC: 15%), male sibling (120C A: 11.5%) -female sibling (120C: A: 11.5%), and male-male siblings (131TC: 11.5). The homological analysis of female-female and male-female siblings showed 11 variants on 143bp HVS-1 mtDNA D-Loop (nt 16259-16410). The highest variants in female-female siblings were: 16387AG, 16387AC: 15%; male sibling-female sibling: 16393CT, 16393CA: 11.5%; while the homological analysis of male-male siblings showed 13 variants with the highest percentage: 16367AG, 16367AC: 11%.