The expression of the mitochondrial transcription factor A (TFAM) gene has a marked impact on mitochondrial biogenesis. It is suggested to play a critical role in lipogenesis, including the deposition of marbling fat in cattle. Bali cattle (Bos javanicus) are known for their lean meat but exhibit poor marbling scores. This study aimed to investigate the polymorphism of c.-1221A>C and novel SNPs in the promoter region of the TFAM gene in Bali cattle. A total of 245 Bali cattle individuals were included in this study. Three Bali, three Wagyu, and one Limousin cattle were used for sequencing analysis. Genomic DNA was extracted either from blood or sperm for genotyping and variant analysis. The c.-1221A>C SNP was genotyped by PCR-RFLP, and the novel SNPs of the TFAM gene promoter region were identified by sequencing analysis. Our results revealed that the c.-1221A>C SNP was found to be monomorphic, with the presence of a fixed A allele, making it unsuitable for further association study in Bali cattle. However, the study successfully identified four novel SNPs and one novel DNP found in the promoter region of the TFAM gene. Notably, one double nucleotide polymorphism (DNP) of g.[45690945C>T;45690946A>G] was unique in Bali cattle compared to other breeds. In addition, only the c.-911T>A SNP was found heterozygous within the Bali cattle samples. These SNPs provide valuable insights into the genetic diversity of the TFAM gene in Bali cattle and serve as a basis for future investigations on the distinctiveness of Bali cattle, especially in the efforts of enhancing the quality of Bali beef.