The 2016 World Health Organization classification introduced a number of genes with somatic mutations and a category for germline predisposition syndromes in myeloid neoplasms. We have designed a comprehensive next-generation sequencing assay to detect somatic mutations, translocations, and germline mutations in a single assay and have evaluated its clinical utility in patients with myeloid neoplasms. Extensive and specified bioinformatics analyses were undertaken to detect single nucleotide variations, FLT3 internal tandem duplication, genic copy number variations, and chromosomal copy number variations. This enabled us to maximize the clinical utility of the assay, and we concluded that, as a single assay, it can be a good supplement for many conventional tests, including Sanger sequencing, RT-PCR, and cytogenetics. Of note, we found that 8.4-11.6% of patients with acute myeloid leukemia and 12.9% of patients with myeloproliferative neoplasms had germline mutations, and most were heterozygous carriers for autosomal recessive marrow failure syndromes. These patients often did not respond to standard chemotherapy, suggesting that germline predisposition may have distinct and significant clinical implications.