In this study, the influence of the β-lactoglobulin, prolactin and αS1-casein variants on milk production traits of three Portuguese sheep breeds, including Serra da Estrela, White Merino and Black Merino, was analyzed. The genetic variants of each marker were identified using PCR-RFLP. β-Lactoglobulin genotype AA was associated with lower milk yield in Serra da Estrela and Merino ewes. This marker also affected milk fat content in Serra da Estrela and protein content in Merino. First results regarding the influence of the sheep prolactin gene on milk production traits are reported. In the Serra da Estrela breed, ewes carrying prolactin genotype AA were significantly associated with lower milk yield, but this influence of prolactin genotypes on milk yield was not detected in Merino. Prolactin genotypes were also significantly associated with milk fat and protein contents in the Serra da Estrela breed. A suggestive effect of the αS1-casein locus on milk yield was detected in Serra da Estrela, but no associations were found between the variants of this marker with milk fat and protein content.