It has been proposed that neurodegenerative diseases are associated with the environmental neurotoxin β-N-methylamino-L-alanine (BMAA). In a rat model, exposure to BMAA from infancy resulted in preferential absorption in the hippocampus, which led to cell death, mineralization, and astrogliosis in addition to impairments in learning and memory in maturity. Furthermore, in the adult hippocampal region's cornus ammonis 1 (CA1) region, exposure to neonates enhanced protein ubiquitination, suggesting that BMAA may cause protein aggregation. With excellent chemical selectivity and great spatial resolution, time-of flight secondary ion mass spectrometry (ToF-SIMS) based imaging is a potent tool for the spatial profiling of tiny molecular weight molecules in biological tissues. This study sought to characterize the neurochemical alterations in the hippocampal regions of six-month-old rats that had received neonatal (postnatal days 9–10) BMAA treatment. Anatomical regions of interest were identified by performing multivariate data analysis of whole section ToF-SIMS images, taking into account the patterns of chemical distribution. Long-term alterations brought on by BMAA were discovered by additional examination of spectral data collected from the indicated anatomical locations, such as the dentate gyrus (DG) and CA1. Phospholipid depletion in the DG and elevated amounts of protein fragments and phospholipids in the histopathologically damaged CA1 area were noted. Furthermore, in the histopathologically changed CA1, high resolution SIMS imaging showed a particular localization of potassium, protein signals, and phosphatidylcholine lipids. These results show that ToF-SIMS based imaging is a potent method for examining biochemical alterations in situ and could be a useful tool for examining brain disease caused by neurotoxins.