This study aimed to characterize the features and partial variation in the D-loop region of mtDNA in Biromaru chickens and to evaluate their genetic relationships with other Indonesian local chicken populations using nucleotide diversity. A total of 46 male and 54 female Biromaru chickens, uniformly aged (grower phase) were collected from Loru Village, Sigi Biromaru District, Sigi Regency, for phenotypic analysis. Additionally, 10 blood samples were taken for DNA extraction, amplification, genotyping, and sequencing. The mitochondrial D-loop region was specifically amplified using the primers: Forward (F): 5’-AGG ACT ACG GCT TGA AAA GC-3’ and Reverse (R): 5’-ATG TGC CTG ACC GAG GAA CCA G-3’. The sequencing results from 10 samples were compared with 17 reference D-loop sequences of Gallus gallus obtained from the GenBank database of NCBI. The genetic distance between Biromaru chickens and other Indonesian local chicken populations was relatively low (<0.099). Phylogenetic analysis revealed that Biromaru chickens grouped within a single cluster along with domestic chickens (Gallus gallus domesticus) and other Indonesian local breeds. The Biromaru chickens showed considerable phenotypic variation. The haplotype network identified ten haplotypes among the Biromaru chicken population. In conclusion, Biromaru chickens exhibit diverse phenotypic traits and share a close genetic relationship with other local chicken populations, although some form distinct sub-clusters, indicating genetic differentiation from other regional local chickens. Therefore, Biromaru chickens can serve as a valuable local genetic resource for conservation and development programs and provide essential baseline information for future genetic research.