The projection of retinal ganglion cell axons to the dorsal lateral geniculate nucleus of the thalamus (dLGN) is organized into eye-specific layers, which are macroscopic structures that reflect the bulk organization of thousands of axons. The processes that underlie the formation of these layers is the focus of research in several laboratories. The recent advent of fluorescently tagged tracers allows for the simultaneous visualization of axons from both eyes in the same dLGN section and therefore the analysis of axonal segregation patterns. However, the techniques traditionally used to quantify eye-specific segregation are far from standardized. Here we present an analysis method that objectively quantifies the extent of segregation. We apply this analyses to dLGN images from mice with normal retinogeniculate projection patterns and genetically altered mice with dramatically altered projection patterns. In addition, we compare dLGN images acquired at different optical resolutions to measure the spatial scale over which we can determine segregation unambiguously.