Within the past few years, the invention of next-generation sequencing has revealed several new genes associated with tumor formation and development, for example DNMT3a. This gene is an independent prognostic factor for acute myeloid leukemia (AML). The objective of this study was to analyze the DNMT3a mutation in childhood AML in a single center. PCR amplification of the entire coding region of DNMT3a was performed using 23 overlapping primer pairs in 57 patients who were diagnosed in Blood Disease Hospital of Chinese Academy of Medical Sciences, then the directly sequencing was underwent. The results showed that no DNMT3a mutation was found in these patients including the hotspot R882. But AML1/ETO mutation was found in 10 patients, CBFB/MYH11 mutation in 3 patients, PML/RARa mutation in 13 patients, FLT3/ITD mutation in 5 patients, FLT3/TKD mutation in 1 patient, PML/RARa and FLT3/TKD mutation coexisted in 2 patients. It is concluded that DNMT3a mutations are rare in childhood AML, and different mechanisms of myeloid leukemogenesis between childhood and adults maybe involved.