The molecular phenotypic heterogeneity of mast cells (MCs) makes them attractive as potential therapeutic targets in anti-cancer adjuvant therapy. Mast cell aggregations observed in tumors suggested their involvement in tumor pathogenesis. Despite several studies using mast cell tryptase, MCs’ involvement in the progression of prostate tumors has not been demonstrated. The aim of our study was to identify and quantify the phenotypic heterogeneity of MCs in prostate lesions. Our study included 7 cases of normal prostate, 25 cases of benign epithelial hyperplasia and 64 cases of prostate carcinoma. MCs were immunohistochemically assessed using three markers: tryptase, chymase and CD117. Two immunophenotypes of MCs were identified in benign lesions: tryptase+/CD117+/chymase– and tryptase–/chymase+/CD117+, located in peritumoral areas. Intratumoral MC phenotype of malignant lesions was characterized by tryptase+/chymase+/CD117+, while in the peritumoral areas three different MCs phenotypes were identified: tryptase+/chymase+/CD117-, tryptase+/CD117+/chymase– and chymase+/CD117+/tryptase–. Our results suggest the correlation of chymase positive MCs of the peritumoral areas and CD117 positive MCs of the intratumoral areas with tumor grade.