A novel primer design method is described for site-directed fragment deletion, insertion, and substitution by PCR that is based on inverse PCR using a single pair of partially complementary primers. This method allowed insertion or substitution of fragments up to 27 bp and deletion of fragments up to 105 bp with screening of candidate colonies complete within 24h. To demonstrate the principle behind this new mutagenesis strategy, a series of deletions, insertions, and substitutions were introduced into the capsid protein gene of satellite panicum mosaic virus (SPMV). This method can potentially facilitate high-throughput gene engineering, structure-function analyses, and library construction to study virus-host interactions.