The magnesium ion is one of the divalent cations that have an important role in chromosome condensation. Research about the role of Mg2 + has been conducted on the chromosomes from various types of cells, especially human cancer cell lines. Nonetheless, no research has been conducted on normal human cells, such as blood cells and lymphocytes. Therefore, a study to evaluate the effect of Mg2+ on chromosome structure in normal cells is required. This study aims to assess the effects of Mg2+ on chromosome structure in lymphocytes using GTL-banding and karyotyping. Metaphase chromosomes were obtained by culturing blood cells, followed by banding using Giemsa and Leishman stain. Mg2+ role was assessed using XBE5 solution (5 mM Mg2+) as control, XBE solution (0 mM Mg2+), and 1 mM EDTA solution as a cation chelator. The chromosome value from each treatment was evaluated using Quality Assessment from the Association for Clinical Cytogenetics according to the International System for Human Cytogenomics Nomenclature (ISCN). The result shows that the control chromosome had a compact structure with a clear banding pattern and bold band intensity, while those treated with XBE and EDTA had a less compact and fibrous structure with an unclear banding pattern and thin band intensity. In addition, the average chromosome score±SD of the XBE (4.389 ± 0.607) and EDTA-treated (4.611 ± 0.607) chromosomes were higher than those of the control (4.222 ± 0.427). These data indicated that Mg2+ is essential in maintaining the structure of the human lymphocyte chromosome. © 2025 Elsevier Ltd