The JAK1/JAK2 inhibitor Ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in two phase III studies against placebo (COMFORT I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% CI:0.30-1.08) versus best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with myelofibrosis treated with ruxolitinib.