Mutational profiling using a custom 43-gene next-generation sequencing panel revealed that patients with mutated DNMT3A or EZH2, or an increase in TET2 VAF and lower TP53 VAF showed a higher overall response. NRAS and TP53 variants were associated with shorter overall survival (OS), whereas only mutated BCOR was associated with a shorter relapse-free survival (RFS). Subgroup analyses of OS according to biological and genomic characteristics showed that patients with low-intermediate cytogenetic risk and mutated NRAS benefited from azacytidine therapy and patients with mutated TP53 showed a better RFS in the azacytidine arm. In conclusion, differential mutational profiling might anticipate the outcomes of first-line treatment choices (AZA or FLUGA) in older patients with AML. We sought to predict treatment responses and outcomes in older patients with newly diagnosed acute myeloid leukemia (AML) from our FLUGAZA phase III clinical trial (PETHEMA group) based on mutational status, comparing azacytidine (AZA) with fludarabine plus low-dose cytarabine (FLUGA). Mutational profiling using a custom 43-gene next-generation sequencing panel revealed differences in profiles between older and younger patients, and several prognostic markers that were useful in young patients were ineffective in older patients. We examined the associations between variables and overall responses at the end of the third cycle. Patients with mutated DNMT3A or EZH2 were shown to benefit from azacytidine in the treatment-adjusted subgroup analysis. An analysis of the associations with tumor burden using variant allele frequency (VAF) quantification showed that a higher overall response was associated with an increase in TET2 VAF (odds ratio (OR), 1.014; p = 0.030) and lower TP53 VAF (OR, 0.981; p = 0.003). In the treatment-adjusted multivariate survival analyses, only the NRAS (hazard ratio (HR), 1.9, p = 0.005) and TP53 (HR, 2.6, p = 9.8 × 10 −7) variants were associated with shorter overall survival (OS), whereas only mutated BCOR (HR, 3.6, p = 0.0003) was associated with a shorter relapse-free survival (RFS). Subgroup analyses of OS according to biological and genomic characteristics showed that patients with low-intermediate cytogenetic risk (HR, 1.51, p = 0.045) and mutated NRAS (HR, 3.66, p = 0.047) benefited from azacytidine therapy. In the subgroup analyses, patients with mutated TP53 (HR, 4.71, p = 0.009) showed a better RFS in the azacytidine arm. In conclusion, differential mutational profiling might anticipate the outcomes of first-line treatment choices (AZA or FLUGA) in older patients with AML. The study is registered at ClinicalTrials.gov as NCT02319135