Structural, functional, and molecular alterations in excitatory spines are a common hallmark of many neurodevelopmental disorders including intellectual disability and autism. Here, we describe an optimized methodology, based on combined use of DiI and immunofluorescence, for rapid and sensitive characterization of the structure and composition of spines in native brain tissue. We successfully demonstrate the applicability of this approach by examining the properties of hippocampal spines in juvenile KO mice, a mouse model of Fragile X Syndrome. We find that mutant mice display pervasive dysgenesis of spines evidenced by an overabundance of both abnormally elongated thin spines and cup-shaped spines, in combination with reduced density of mushroom spines. We further find that mushroom spines expressing the actin-binding protein Synaptopodin-a marker for spine apparatus-are more prevalent in mutant mice. Previous work identified spines with Synaptopodin/spine apparatus as the locus of mGluR-LTD, which is abnormally elevated in KO mice. Altogether, our data suggest this enhancement may be linked to the preponderance of this subset of spines in the mutant. Overall, these findings demonstrate the sensitivity and versatility of the optimized methodology by uncovering a novel facet of spine dysgenesis in KO mice.