To find a better condition for KIT immunostaining, five antibodies against KIT were compared, including a widely used polyclonal antibody (pAb) A4502, three mouse-derived mAb (MMA; T595, 1DC3, K45), and a newly developed rabbit-derived mAb (RabMA; Y145). Twenty-three gastrointestinal stromal tumors (GIST) were stained, including five KIT-weak or -negative GIST with PDGFRA gene mutations from a previous report, six Ewing/malignant primitive peripheral neuroectodermal tumor, six malignant melanomas, two neuroblastomas, six seminomas, seven thymic carcinoma and seven small cell carcinomas of the lung as KIT-expressing tumors, and four leiomyomas, six leiomyosarcomas, five gastric schwannomas, four solitary fibrous tumors, one inflammatory fibroid polyp and six desmoid tumors as KIT-non-expressing tumors. The positive rates of RabMA Y145 in KIT-expressing tumors were almost equal to pAb A4502, whereas those of three MMA had lower rates. MMA T595 was positive for mast cells, but negative for interstitial cells of Cajal and some GIST. None of the KIT-non-expressing tumors was positive for Y145, whereas some leiomyosarcomas and desmoid tumors were positive for A4502. At present, pAb A4502 or RabMA Y145 seems to be suitable for KIT immunostaining in formalin-fixed paraffin-embedded tumor specimens, especially in the differential diagnosis of GIST from other mesenchymal tumors.