The animal selection with favourable phenotypes of the past has been, currently, replaced by the genotype selection on quantitative traits, assisted by the expanding molecular techniques in the context of livestock improvement. In this study, the c. 112T>C polymorphism in exon II of beta-lactoglobulin (beta-LG) gene was investigated in Karagouniko and Chios sheep breeds by using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP), and possible associations with milk traits were examined. In total, 125 blood DNA samples were isolated for PCR-RFLP analysis and the respective 217 milk samples' composition profile was obtained. The goodness of fit test to Hardy-Weinberg equilibrium (HWE) for beta-LG genotypes was estimated and associations found between beta-LG genotypes and raw milk composition. Two alleles and three genotypes were observed (AA, AB and BB) in both breeds, and Chios breed significantly deviated (P <= 0.05) from Hardy-Weinberg equilibrium (HWE). Conclusively, linear mixed model analysis on samples, from both breeds collectively, showed significant effects of beta-LG genotype on lactose percentage and somatic cell count (SCC), lactation stage on daily milk yield and protein, while the breed effect was significant only on daily milk yield.