Detection of activating c-kit mutation D816 V is one of five criteria for the diagnosis of systemic mastocytosis (SM). The aims of this study were to (I) establish molecular methods for the detection of these mutations in paraffin-embedded biopsies, (II) determine the frequency of these mutations in mastocytoses and control tissues, (III) determine the frequency of these mutations in laser-microdissected lesional and nonlesional mast cells (MC), and (IV) investigate these matutions as a marker for clonality in cases with SM and associated clonal hematologic non-mast cell lineage diseases (SM-AHNMD). Formalin-fixed and paraffin-embedded biopsies of 48 patients with cutaneous mastocytosis (CM), 55 cases with various forms of SM, and 239 controls were investigated by PNA-mediated PCR-clamping. In addition, nested PCR amplified DNA of pooled microdissected single mast cells (MC) was investigated by melting point analysis. Activating c-kit mutation codon 816 mutations were detected in 38 % (18/48) of CM, in 91% (50/55) of SM, in 5 % (2/39) of MC hyperplasia and in none of 200 hematologic non-MC neoplasias. c-kit mutations were detected significantly more frequent in lesional MC as compared to non-lesional MC (p = 0,003). In 6/15 (40 %) cases with SM-AHNMD the same c-kit mutations were detected in microdissected MC and AHNMD cells. This study underlines the concept of the actual WHO classification of mastocytoses. By establishing methods for the detection of c-kit codon 816 mutations in paraffin-embedded tissues, the pathologist holds a central position in the diagnosis of systemic mastocytoses.