ABSTRACT: A myeloid neoplasm-relevant 27-gene panel was used for next-generation sequencing of bone marrow or whole blood DNA in 182 patients with primary myelofibrosis (PMF). DNA sequence variants/mutations other than JAK2/CALR/MPL were detected in 147 patients (81%), with the most frequent being ASXL1 (36%), TET2 (18%), SRSF2 (18%), and U2AF1 (16%); furthermore, 35%, 26%, 10%, and 9% of the patients harbored 1, 2, 3, or 4 or more such variants/mutations, respectively. Adverse variants/mutations were identified by age-adjusted multivariable analysis of impact on overall survival or leukemia-free survival and included ASXL1, SRSF2, CBL, KIT, RUNX1, SH2B3, and CEBPA; their combined prevalence was 56%. Adverse variants/mutations were associated with inferior overall survival (median, 3.6 vs 8.5 years; P < .001) and leukemia-free survival (7-year risk, 25% vs 4%; P < .001), and the effect on survival was independent of both the Dynamic International Prognostic Scoring System Plus and JAK2/CALR/MPL mutational status, with respective hazard ratios of 2.0 (95% confidence interval [CI], 1.3-3.1) and 2.9 (95% CI, 1.9-4.4). Additional prognostic information was obtained by considering the number of adverse variants/mutations; median survivals in patients with zero (n = 80), 1 or 2 (n = 93), or 3 or more (n = 9) adverse variants/mutations were 8.5, 4, and 0.7 years, respectively (P < .001). Additional data were obtained on pattern of mutation co-segregation and phenotypic correlation, including significant associations between U2AF1 and JAK2 mutations (P = .04) and U2AF1 mutations and anemia (P = .003) and thrombocytopenia (P = .006). We conclude that DNA variants/mutations other than JAK2/CALR/MPL are prevalent in PMF and are qualitatively and quantitatively relevant in predicting overall and leukemia-free survival.