Activating mutations of KIT play an important role in the pathophysiology of several human malignancies, including acute myeloid leukemia. Activated KIT kinase is therefore a promising molecular target for the treatment of many malignancies harboring KIT activation. Here we examined the potency of a novel KIT inhibitor KI-328 against different types of mutant KIT kinases recurrently identified in AML. KI-328 shows selective potency against KIT kinase for the in vitro kinase assay, and inhibits the growth of wild-type (Wt)- and mutant-KIT-expressing cells, while it has little potency against D816V-KIT. Comparable analysis of several potent KIT inhibitors regarding growth inhibitory effects on a variety of mutant-KIT-expressing cells revealed that multi-kinase inhibitors have the same potency against D816V-KIT as other mutant KITs; however, the predominant potency against D816V-KIT was observed in heat shock protein 90 (HSP90) inhibitors. Furthermore, HSP90 inhibitors suppress the growth of D816V-KIT-expressing cells at the concentration at which the growth of other mutant-KIT-expressing cells is not affected. These results collectively indicated that potent KIT inhibitors have different potency against the type of mutant KIT kinases. Therefore, KIT inhibitors are required to validate potency against several types of mutant KIT kinases for the clinical development.