Type IV collagens are the most abundant proteins in basement membranes. Distinct genes encode each of six isoforms, alpha1(IV) through alpha6(IV), which assemble into one of three characteristic heterotrimers. Disease-causing mutations in each of the six genes are identified in humans or mice and frequently include diverse ocular pathogenesis that encompass common congenital and progressive blinding diseases, such as optic nerve hypoplasia, glaucoma, and retinal degeneration. Understanding where and when collagen IV molecules are expressed is important because it defines limits for the location and timing of primary pathogenesis. Although localization of collagen IV isoforms in developed human eyes is known, the spatial and temporal distribution of type IV collagens throughout ocular development has not been determined in humans or in mice. Here, we use isoform-specific monoclonal antibodies to systematically reveal the localization of all six collagen IV isoforms in developing mouse eyes. We found that alpha1(IV) and alpha2(IV) always co-localized and were ubiquitously expressed throughout development. alpha3(IV) and alpha4(IV) also always co-localized but in a much more spatially and temporally specific manner than alpha1(IV) and alpha2(IV). alpha5(IV) co-localized both with alpha3(IV)/alpha4(IV), and with alpha6(IV), consistent with alpha5(IV) involvement in two distinct heterotrimers. alpha5(IV) was present in all basement membranes except those of the vasculature. alpha6(IV) was not detected in vasculature or in Bruch's membrane, indicating that alpha5(IV) in Bruch's membrane is part of the alpha3alpha4alpha5 heterotrimer. This comprehensive analysis defines the spatial and temporal distribution of type IV collagen isoforms in the developing eye, and will contribute to understanding the mechanisms underlying collagen IV-related ocular diseases that collectively lead to blindness in millions of people worldwide.