The differences between renneting characteristics of raw milk samples from different origins (bovine, ovine, caprine, buffalo) were investigated by protein surface hydrophobicity approach. 8-Anilinonaphthalene-1-sulfonic acid (ANS) binding method was used to evaluate surface hydrophobicity of raw milk samples and rennet precipitates. The following surface hydrophobicity parameters were calculated: number of surface hydrophobic sites ( F _max), dissociation constant of the fluorescent ANS–protein complex ( K _d), binding affinity of ANS to protein surface (1/ K _d), the average tightness of binding of ANS to the protein ( F _max/ K _d), turnover number ( k _cat), and protein surface hydrophobicity index (PSHI). The number of hydrophobic sites on the protein surface was found to be highest in cow milk, whereas ovine milk samples had the lowest number of hydrophobic sites and binding affinity to ANS. Protein content was not found directly related to the number of surface hydrophobic sites. The binding affinity of the proteins to ANS was greater in buffalo milk. PSHI was found to be the highest for bovine milk and the lowest for ovine milk. Renneting period was interpreted in two phases (enzymatic phase and flocculation phase) for each origin via ANS partition curves of rennet precipitates. Same trends between bovine–ovine and caprine–buffalo milks were observed during renneting. Buffalo milk completed both of two phases and total renneting period significantly earlier than the milks from the other origins. The hydrophobic parameters of proteins were found to play a key role on coagulation properties.