Phyllodes tumors (PTs) are rare breast tumors showing overlapping features with fibroadenomas (FAs). Diagnosis on small biopsies is challenging. New diagnostic markers are needed. Here we evaluated immunohistochemical staining of histone 3 trimethyl-lysine-27 (H3K27me3) as a diagnostic and prognostic marker in a series of PTs. Surgically removed PTs at our institution (September 1990 and July 2022) and control FAs. Tissue micro-arrays (4 cores, 2 mm Ø) stained with H3K27me3, and scored with QuPath-derived H-score. Fisher exact test, Mann-Whitney U-test and chi-squared test used for group comparison. ROC analysis applied to define cutoffs. Cox proportional hazards models were used for assessing disease-free survival (DFS), overall survival (OS), and disease-specific survival (DSS) in PTs. We included 81 patients with PTs and 44 patients with FAs. QuPath-derived H-scores of stromal H3K27me3 were statically significantly lower in PTs than in FAs (p < 0.001). We identified exploratory cutoffs to discriminate FAs from benign and malignant PTs (AUC = 0.78 and 0.73, respectively). No associations between DFS, OS, or DSS and H3K27me3 expression were found. H3K27me3 expression differs between FAs and PTs, indicating potential as diagnostic marker, but it is not predictive for DFS, OS or DSS in PTs. Further validation is needed.