Background Acute megakaryoblastic leukemia (AMKL) without Down syndrome (non-DS-AMKL) usually a worse outcome than DS-AMKL. Acquired trisomy 21(+21) was one of the most common cytogenetic abnormalities in non-DS-AMKL. Knowledge of the difference in the clinical characteristics and prognosis between non-DS-AMKL with +21 and those without +21 is limited. Objective Verify the clinical characteristics and prognosis of non-DS-AMKL with +21. Method We retrospectively analyzed 33 non-DS-AMKL pediatric patients and 118 other types of AML, along with their clinical manifestations, laboratory data, and treatment response. Results Compared with AMKL without +21, AMKL with +21 has a lower platelet count (44.04 & PLUSMN; 5.01G/L) at onset (P > 0.05). Differences in remission rates between AMKL and other types of AML were not significant. Acquired trisomy 8 in AMKL was negatively correlated with the long-term OS rate (P < 0.05), while +21 may not be an impact factor. Compared with the other types of AML, AMKL has a younger onset age (P < 0.05), with a mean of 22.27 months. Anemia, hemorrhage, lymph node enlargement, lower white blood cell, and complex karyotype were more common in AMKL (P < 0.05). AMKL has a longer time interval between onset to diagnosis (53.61 & PLUSMN; 71.15 days) (P < 0.05), and patients with a diagnosis delay & GE;3 months always presented as thrombocytopenia or pancytopenia initially. Conclusions Due to high heterogeneity, high misdiagnosis rate, and myelofibrosis, parts of AMKL may take a long time to be diagnosed, requiring repeated bone marrow punctures. Complex karyotype was common in AMKL. +21 may not be a promising indicator of a poor prognosis.