Phyllodes tumors of the breast are diagnostically and managerially enigmatic, as their malignant potential is difficult to predict based on the standard morphologic criteria. Thus, there is a need for additional markers of biologic potential. Although a number of ancillary tests have been reported, consensus in the literature is lacking. We studied 38 cellular fibroadenomas and phyllodes tumors of various grade (World Health Organization benign, borderline, and malignant) with a panel of immunohistochemical stains (p53, CD117, phospho-Histone3, mdm2, cdk4) and screened 26 of the tumors for mutations across 30 cancer-related genes using PCR and mass-spectrometry based methods. p53 and phospho-Histone3 (mitotic marker) showed increased staining in higher grade phyllodes tumors. CD117, mdm2, and cdk4 showed no difference in expression across different grades of phyllodes tumors. Mutational analysis revealed an S8R substitution in FBX4 (an E3 ubiquitin ligase) in 3 cases: 1 benign and 2 borderline. The S8R substitution seems to be more common in phyllodes tumors (11.5%) as compared with other cancers. FBX4 S8R cases had high cyclin D1 expression, but this finding was not specific. Our data support earlier studies showing that p53 has potential use in pathologic assessment of phyllodes tumors, and we newly characterized phospho-Histone3 for this application. Further studies are needed to characterize the molecular pathogenesis of the phyllodes tumors, as we were unable to identify activating mutations despite screening for a large panel of activating hotspot mutations. The significance of the FBX4 substitution deserves further investigation.