Sialyloligosaccharides (SOS) are bioactive molecules that play an important role in brain development and the increase in immunity in infants. In adults, they act as prebiotics, enhancing protection against microbial pathogens. In the present work, we aimed to analyze the levels of SOS in mature milk, at days 60 and 120 after calving in four cow breeds: Holstein (HO), Simmental × Holstein (SM × HO), Simmental (SM), all fed with total mixed ration (TMR) in intensive production, and Podolica (POD) raised on pasture in an extensive system. The concentrations of SOS (3′-sialyllactose = 3′-SL, 6′-sialyllactose = 6′-SL, 6′-Sialyl-N-acetyllactosamine = 6′-SLN, disialyllactose = DSL, expressed in mg/L) were determined using HPAEC-PAD, a high-performance anion-exchange chromatography with pulsed amperometric detection. Results showed both breed and lactation effects. The contents of 3′-SL, 6′-SL, 6′-SLN, and DSL were higher at 60 than 120 days (p < 0.001), as well as in POD, as compared to the other breeds (p < 0.001). Furthermore, SM showed a significantly greater level of 3′-SL than HO (p < 0.001), as well as a significantly higher level of 6′-SLN in SM than HO (p < 0.001) and SM × HO (p < 0.001). Our findings may have implications for several areas of sustainability that might be used in the cattle management system.