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This study addresses the challenges in diagnosing clinically difficult blood groups by employing genotyping and sequencing techniques to enhance blood supply accuracy. Two patients with challenging blood groups underwent serological testing and molecular diagnostics using fluorescent PCR typing and Sanger sequencing. Patient 1 showed a discrepancy in serological typing, indicating a B subgroup confirmed by a B\/O\\*1 genotype and a 721C&gt;T mutation in the ABO gene Exon 7, establishing the Bw. 03\/O.01.01 genotype. Patient 2, with negative Rh antigen detection and typed as CcDEe, was diagnosed with a Rh-null phenotype due to a homozygous frameshift mutation (c.732delC) in the RHAG (Rh-Associated Glycoprotein) gene Exon 5. Integrating serological screening with genotyping and sequencing technologies is recommended for accurately identifying challenging blood groups. 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