Melatonin (MT) is an important bioactive functional component of milk, where its content and biosynthetic mechanism are of considerable relevance to the quality regulation of dairy buffalo milk. This study aimed to investigate the variation patterns and biosynthetic mechanisms of melatonin in milk from dairy buffaloes. A total of 125 buffalo milk samples were collected, and MT content was determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS). MT secretion patterns were evaluated according to breed, lactation, parity, and milking time (light exposure). In addition, whole-genome sequencing (WGS) data were used to screen for single-nucleotide polymorphisms (SNPs), which are significantly associated with melatonin biosynthetic enzyme genes. The results showed that breed, milking time, and lactation were the major factors affecting MT content in buffalo milk. Based on the WGS data, two SNPs significantly associated with MT secretion and synthesis were identified within the aralkylamine N-acetyltransferase (AANAT) region, among which the GG genotype at 3-8019407 and the CC genotype at 3-8019446 were associated with higher MT content. The MT content for both wild-type homozygotes was 59.54 pg/mL, which was higher than that of heterozygotes (39.12 pg/mL) (p < 0.05). With the exception of SNP2 and SNP8, all other SNPs were in Hardy–Weinberg equilibrium. These findings will serve as a reference for the subsequent selection and identification of breeding materials for high MT dairy buffalo, as well as for the development of high MT buffalo milk products.