Recently, c-kit mutations have been reported as a novel adverse prognostic factor of acute myeloid leukemia with t(8;21)(q22;q22) translocation (t(8;21) AML). However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive mutation detection method known as mutation-biased PCR (MB-PCR) and investigated the relationship between c-kit mutations and prognosis. When c-kit mutations were analyzed for 26 cases of t(8;21) AML using the direct sequence (DS) and MB-PCR, the latter had a much higher detection rate of c-kit mutations at initial presentation (DS 5/26(19.2%) vs MB-PCR 12/26(46.2%)). Interestingly for the three cases, in which c-kit mutations were observed only at relapse with the DS, c-kit mutations were detected at initial presentation using the MB-PCR. This result suggests that a minor leukemia clone with c-kit mutations have resistance to treatment and are involved in relapse. In univariate analyses, the presence of a c-kit mutation using DS was not an adverse prognostic factor (P = 0.355), but was a factor when using MB-PCR (P = 0.014). The presence of c-kit mutations with MB-PCR was also an independent adverse prognostic factor by multivariate analyses (P = 0.006). We conclude that sensitivity of c-kit mutation detection method is important to predict prognosis for t(8;21) AML.