Objective: To explore the application and discovery of genotyping, gene sequencing, and gene expression analysis in the determination of ABO blood group subtypes and antigen expression abnormalities in hematological malignancies patients. Methods: From June 2019 to May 2020, three clinical cases were found with forward and reverse ABO typing discrepancy or atypical serologic agglutination pattern in the laboratory and blood transfusion department of Hebei Yanda Ludaopei Hospital were selected. Sequence-specific primer PCR (PCR-SSP) and Sanger sequencing of ABO gene coding regions were performed to determine the ABO genotypes, and whole transcriptome sequencing was used to analyze ABO and FUT1 gene expression levels. Results: A 12-year-old female acute lymphoblastic leukemia patient was determined as O.01.02 and BA.04 sub-genotype, corresponding to the serological B(A) subtype, and her ABO gene expression was normal (354.80). A 41-year-old female acute myeloid leukemia patient was determined as A1.02 and B.01 genotype, corresponding to the serological A(1)B phenotype, and her ABO gene expression was significantly reduced (45.70). A 42-year-old male with myelodysplastic syndrome and myelofibrosis was determined as A1.02 and A2.05 sub-genotype, corresponding to the serological A(1) and A(2) phenotype, respectively, and his ABO expression was negative. FUT1 expression was in the normal range in all three cases. The clinical blood product infusion strategy was formulated according to the genotype and the corresponding immunological subtype, and no significant transfusion-related adverse reactions occurred. Conclusion: Blood group sub-genotypes or aberrant gene expression can lead to ambiguities in serological blood group determination in hematological malignancies patients. ABO genotyping and gene expression analysis can help in this scenario and escort blood product infusion safety.