We have previously reported the possible segregation of major gene(s) for milk yield and fat corrected milk (FCM) in Chios sheep breed. In this study we demonstrate the association of an SNP marker, within the last exon of the Acetyl-CoA acyltransferase 2 (ACAA2) gene, with milk yield and FCM in the same breed. Moreover, we investigate the effect of this SNP marker along with other microsatellite markers. Fifteen microsatellite markers (SSR) on 13 chromosomes were genotyped on 198 ewes of the Chios breed. The association analysis revealed significant association (P<0.03) between one microsatellite marker (OarFCB128) with milk yield, fat content and FCM while two other microsatellite markers (MAF214 and OarFCB304) were found to be marginally significant (P<0.09). All 3 microsatellite markers are mapped on genomic areas that have been previously reported to contain Quantitative Traits Loci (QTL) affecting milk yield in other sheep breeds. Inclusion of 318 ewes SNP marker genotypes of the ACAA2 gene in the association analysis resulted in significant association of the SNP marker with milk yield traits but no significant association for the microsatellite marker(s). Further investigation on possible interaction(s) between these loci with the SNP mutation of the ACAA2 gene is needed. [ABSTRACT FROM AUTHOR]