Genetic analysis of von Willebrand disease by von Willebrand factor gene sequencing has not yet become routine practice. Nevertheless, the prospects for molecular diagnosis have changed dramatically in recent years with the unveiling of next-generation sequencing platforms. With the goal of applying this technology to von Willebrand disease, we designed a strategy for von Willebrand factor gene enrichment and multiplexing based on short PCRs. 40 patients were simultaneously analyzed enabling the identification of 43 mutations, including 36 substitutions, 2 intronic splice site mutations, 2 indels, and 3 deletions. Pooling patient genomic DNA before PCR enrichment, indexing samples with barcode tags, and re-sequencing on the next-generation sequencing instrument, at least 350 patients and relatives per run can be simultaneously analyzed in a fast, inexpensive manner. This is one the first reports in which this technology has shown feasibility for large-scale mutation screening by single gene re-sequencing.