Fragile X syndrome (FXS), caused by inactivation of Fmr1 gene, is the most common cause of intellectual disability and autism spectrum disorder. Fmr1 knockout (Fmr1KO) mice display a disorganization of perineuronal nets (PNNs), specialized forms of extracellular matrix which form around neurons during postnatal development in many brain areas. These nets are preferentially established around Parvalbumin-positive GABAergic interneurons (PVIs), and their presence reduces brain plasticity. PNN and PVI expression is altered in various neuropsychiatric disorders and in some cases their manipulation has been able to reverse physiological and behavioral deficits. Despite that, little is known about their distribution and a systematic study of PNN expression in FXS mice has never been conducted. Here, we performed a brain-wide analysis of PNNs in Fmr1KO and C57 wild type (WT) mice to extend knowledge of FXS-dependent PNN and PVI alterations affecting different brain areas. To this aim we developed a customized method for whole-brain 3D reconstruction of histologically stained sections followed by high-throughput automated image analysis. We found a general reduction of PNN and PVI expression in FXS mice compared to WT, greatly affecting cortex, amygdala, hippocampus and striatum. Focusing on isocortex, we observed a strong decrease of PNNs in FXS mice, especially in deeper cortical layers. Moreover, primary areas were more heavily affected by PNN decrease than the associative ones. This study enabled to dissect whole-brain distribution of PNNs and PVIs in the FXS model, paving the way to identify specific brain areas that could be therapeutically targeted in FXS.