Systemic-to-pulmonary shunt malfunction contributes to morbidity in children with complex congenital heart disease after palliative procedure. Neointimal hyperplasia might play a role in the pathogenesis increasing risk for shunt obstruction. The aim was to evaluate the role of epidermal growth factor receptor (EGFR) and matrix-metalloproteinase 9 (MMP-9) in the formation of neointimal within shunts. Immunohistochemistry was performed with anti-EGFR and anti-MMP-9 on shunts removed at follow-up palliative or corrective procedure. Whole-genome single-nucleotide polymorphisms genotyping was performed on DNA extracted from patients´ blood samples and allele frequencies were compared between the group of patients with shunts displaying severe stenosis (≥ 40% of lumen) and the remaining group. Immunohistochemistry detected EGFR and MMP-9 in 24 of 31 shunts, located mainly in the luminal area. Cross-sectional area of EGFR and MMP-9 measured in median 0.19 mm2(IQR 0.1–0.3 mm2) and 0.04 mm2(IQR 0.03–0.09 mm2), respectively, and correlated positively with the area of neointimal measured on histology (r= 0.729,p< 0.001 andr= 0.0479,p= 0.018, respectively). There was a trend of inverse correlation between the dose of acetylsalicylic acid and the degree of EGFR, but not MMP-9, expression within neointima. Certain alleles in epidermal growth factor (EGF) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were associated with increased stenosis and neointimal hyperplasia within shunts. EGFR and MMP-9 contribute to neointimal proliferation in SP shunts of children with complex cyanotic heart disease. SP shunts from patients carrying certain risk alleles in the genes encoding for EGF and TIMP-1 displayed increased neointima.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00335-023-09982-3.