Mutations in JAK2, MPL and CALR genes have been identified in the majority of myeloproliferative neoplasm (MPN) patients, and patients negative for these three mutations are the so-called triple-negative (TN) MPN. In this study, we examined the mutational profiles of 16 triple-negative MPN patients including 7 essential thrombocythemia (ET), 1 primary myelofibrosis and 8 polycythemia vera (PV). Targeted next-generation sequencing was performed using the ACTOnco Comprehensive Cancer Panel (Ion AmpliSeq Comprehensive Cancer Panel, Life Technologies) to target all coding exons of 409 cancer-related genes. Overall, 30 nonsynonymous somatic mutations were detected in 12 (75%) patients with a range of 1-5 mutations per sample. Notably, one ET patient was found to have JAK2V617F and KITP551L mutations at very low allele frequency. One MPLP70L and 1 MPLM602T mutations were identified each in 1 ET and 1 PV, respectively. Other recurrent mutations were also identified including KMT2C, KMT2D, IRS2, SYNE1, PDE4DIP, SETD2, ATM, TNFAIP3 and CCND2. In addition, germline mutations were also found in some cancer-related genes. Copy number changes were rare in this cohort of TN MPNs. In conclusion, both somatic and germline mutations can be detected in TN MPN patients.