Background and Aim: Milk physicochemical properties play essential role in the milk processing industry, which are moderately to highly affected by genetic factors. This study aimed to evaluate the association between single nucleotide polymorphisms in POU class 1 homeobox 1 (POU1F1) and the physicochemical properties of milk in high-producing Holstein Friesian (HF) cows. Materials and Methods: A total of 149 high-producing dairy cows from PT Ultra Peternakan Bandung Selatan was included in this study. The physicochemical properties of milk, including density, freezing point, pH, lactose, solid non-fat, protein, and ash content, were determined. Moreover, three polymorphisms within the exon regions of POU1F1 (c.195G>A, c.300G>T, and c.828G>A) were analyzed using polymerase chain reaction-restriction fragment length polymorphism. The association between these polymorphisms and the physicochemical properties of milk was determined using a mixed-effects model analysis, in which the lactation period was used as a covariate. Results: This study found that two polymorphisms, c.195G>A and c.828G>A, significantly affected the pH of fresh milk. Cows with both the GG genotypes c.195G>A and c.828G>A had lower milk pH values than those with the other genotypes. In addition, a non-significant effect of POU1F1 was observed on the other physicochemical properties of milk. Conclusion: Two POU1F1 polymorphisms determined the pH of fresh milk in the Indonesian HF population. These are potential marker candidates for milk pH that directly affect the development of dairy products in the milk processing industry. Keywords: exonic mutation, Holstein Friesian, milk pH, physicochemical properties, POU1F1.