Authenticity of cow, goat, ewe and buffalo milk is a common concern worldwide for true labeling and value assessment. Because of their high economic value, these milk species are frequently adulterated with cow milk. Recently, non-invasive and rapid spectroscopic methods have been preferred instead of DNA and protein based methods. In this context, the goal of the present study was to discriminate cow, goat, ewe and buffalo milk species by using synchronous fluorescence spectroscopy (SFS) method. The principal of the method is based on differences in the types and quantities of fluorophore compounds in milk species. With this aim, SF spectra of milk species were recorded between 250 and 550 nm excitation range with Delta lambda of 20-100 nm, in steps of 10 nm. The data obtained from all Delta lambda = 20-100 nm were used in partial least square discriminant analysis (PLS-DA) in order to classify three groups of samples which are: pure-mixed milk, milk species and type of binary mixture. After determination of the binary mixture type (buffalo-cow; ewe-cow; goat-cow), partial least square (PLS) models were developed for quantification of cow milk in other milk species. Limit of detection values of the method were calculated as 3.52%, 4.06% and 3.52% for goat-cow, ewe-cow and buffalo-cow's milk, respectively. The study demonstrated that SF spectroscopy is a promising tool and has potential to become a rapid and nondestructive analytical technique for identification of milk species.