Chromosomes serve as fundamental units for the transmission of genetic information, and understanding their higher-order structure has been a longstanding research challenge. This review highlights recent advances in plant chromosome structure analysis, focusing on electron microscopy techniques that enable nanoscale visualization of chromatin architecture. Improvements in chromosome preparation, including chromosome isolation and ionic liquid coating, have enhanced the preservation of chromosome structure. Scanning Electron Microscopy (SEM), Focused Ion Beam SEM (FIB-SEM) and High-Voltage Transmission Electron Microscopy (HVTEM) have revealed detailed features of chromatin folding in centromeric and non-centromeric regions. Proteomic studies have identified key chromosomal proteins, such as topoisomerase II and nucleolar proteins, contributing to chromosome condensation and stability. The role of divalent cations and RNA in chromatin compaction are also discussed. Integrating these findings, this review provides an overview of technological advancements and their impact on elucidating chromosome architecture. © 2025 The Authors