Ketoreductases (KRs) are domains in the reductive loops of type I polyketide synthases (PKSs) and are responsible for the majority of stereocenters in reduced polyketides. Although the highly stereoselective reduction of ACP-bound β-ketothioester intermediates by KRs is crucial for the overall functioning of PKSs, the substrate-dependent stereoselectivity of KRs is a factor that is not yet fully understood, especially for KR domains in late PKS modules that act on biosynthetic precursors with complex polyketidic moieties. We present studies on the three KR domains FosKR7, PlmKR6, and EryKR6 from the biosynthetic pathways of fostriecin, phoslactomycin, and erythromycin by in vitro assays using close surrogates of the octaketidic FosKR7 biosynthetic precursor, complex derivatives and a diketide in the form of their biomimetic N-acetylcysteamine thioesters. Supported by molecular modeling, specific interactions of the studied KR domains with the extended polyketide moieties of their natural precursors were identified and correlated to the differences in stereoselectivity observed in the in vitro assays. These results reinforce the importance of the substrate-dependent stereoselectivity of KR domains in PKSs and suggest more detailed experimental and structural studies with isolated KRs and full PKS modules that could ultimately lead to improved results in PKS engineering.