Acute undifferentiated leukemia is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited and it is uncertain if acute undifferentiated leukemia is biologically distinct from acute myeloid leukemia with minimal differentiation, which also shows limited myeloid marker expression and has been reported to have a poor prognosis. We identified 92 cases initially diagnosed as acute undifferentiated leukemia or acute myeloid leukemia with minimal differentiation from pathology databases of nine academic institutions with available diagnostic flow cytometric data, cytogenetic findings, mutational and clinical data. Outcome analysis was performed using Kaplan Meier test for the 53 patients who received induction chemotherapy. Based on cytogenetic abnormalities (N = 30) or history of myelodysplastic syndrome (N = 2), 32 cases were re-classified as acute myeloid leukemia with myelodysplasia related changes. The remaining 24 acute undifferentiated leukemia patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the remaining 30 acute myeloid leukemia with minimal differentiation patients. Compared to acute myeloid leukemia with minimal differentiation, acute undifferentiated leukemia cases were characterized by more frequent mutations in PHF6 (5/15 vs 0/19, p = 0.016) and more frequent expression of TdT on blasts (p = 0.003) while acute myeloid leukemia with minimal differentiation cases had more frequent CD123 expression (p = 0.042). Outcome data showed no difference in overall survival, relapse free survival, or rates of complete remission between acute undifferentiated leukemia and acute myeloid leukemia with minimal differentiation groups (p > 0.05). Acute myeloid leukemia with myelodysplasia-related changes patients showed shorter survival when censoring for bone marrow transplant as compared to acute undifferentiated leukemia (p = 0.03) and acute myeloid leukemia with minimal differentiation (p = 0.002). In this largest series to date, the acute undifferentiated leukemia group shows distinct characteristics from acute myeloid leukemia with minimal differentiation, including more frequent PHF6 mutations and expression of TdT.