Background and Aim: The leptin (LEP) gene plays a pivotal role in regulating growth, metabolism, and fat deposition in cattle. Genetic polymorphisms in this gene can influence phenotypic traits and may serve as molecular markers for selection in breeding programs. However, comprehensive characterization of LEP gene variants in local Indonesian breeds, such as Madura cattle, remains limited. This study aimed to identify novel single-nucleotide polymorphisms (SNPs) within exon 2 of the bovine LEP gene and assess their association with growth traits in Madura cattle. Materials and Methods: Forty-five Madura cows (aged 2–4 years) were phenotypically evaluated for body weight (BW), wither height (WH), body length (BL), chest girth (CG), hip height (HH), head length (HL), and head width (HW). Genomic DNA was extracted and subjected to polymerase chain reaction amplification followed by Sanger sequencing. Detected SNPs were analyzed for genotype and allele frequencies, Hardy–Weinberg equilibrium (HWE), and their associations with growth traits using a general linear model. Results: Three SNPs were identified in exon 2 of the LEP gene: c.126T>C (synonymous), c.148T>C (missense), and a novel missense SNP c.149G>A, resulting in a cysteine to tyrosine substitution at amino acid position 50. The c.149G>A SNP showed polymorphism with three genotypes (GG, GA, AA), and the heterozygous GA genotype had the highest frequency (64.4%). This SNP deviated from HWE (p < 0.05), indicating potential selection pressure or population structure effects. While no statistically significant associations were found between genotypes and growth traits (p > 0.05), the AA genotype showed the highest mean values across most body measurements. Conclusion: Although the novel c.149G>A SNP was not significantly associated with growth traits, its polymorphic nature and descriptive trait patterns suggest it may have biological relevance. Larger-scale studies are recommended to validate its utility as a genetic marker for growth and development in Madura cattle. Keywords: c.149G>A, growth traits, leptin gene, Madura cattle, missense mutation, molecular marker, single-nucleotide polymorphism.