A participatory animal-breeding program was applied to 9 commercial Awassi sheep flocks in Jordan. This study aimed to assess the influence of Beta-lactoglobulin (β-LG), Prolactin (PRL), and Kappa casein (CSN3) genes, genotypes and their interaction on milk production and composition traits of 167 genotyped Awassi ewes via Polymerase Chain Reaction (PCR) followed by sequencing. Allele frequencies for the two variants were 0.42 and 0.58 for β-LG, 0.82 and 0.18 for PRL, and 0.92 and 0.08 for CSN3. No association was found among β-LG and CSN3 polymorphic genotypes with milk production traits. However, ewes with PRL AA genotype showed higher milk production, β-LG AB was associated with lowest fat%, high solid not fat (SNF)%, protein%, and lactose%. β-LG BB was associated with highest milk density. PRL, β-LG, and CSN3 polymorphic genotypes were differentially associated with milk production and component traits. Furthermore, β-LG × PRL interaction showed the highest milk production and fat%; β-LG × PRL recorded the highest SNF%, protein%, lactose%, and milk density, while the PRL × CSN3 had the highest fat% and SNF%. The enhancing effects of these gene interactions can be incorporated in Awassi breeding programs to improve milk production and composition.