We developed a novel method to construct mutagenesis libraries via in situ error-prone PCR. One of our recent PCT patents described a novel method that adding a ligation step mediated by thermostable Thermotoga maritima (Tma) DNA ligase to form the repeated PCR cycles of “denaturation–annealing–elongation–ligation”, which allows exponential amplification of circular DNA. In this method, circular PCR products are generated by using a long dsDNA primer pair, which is designed to carry a selection marker different from the original selection marker of the template plasmid, the template plasmid carrying the original marker is eliminated when transformed host cells are cultured under the new selection pressure. If the product serves as the template for the next round of amplification, accumulation of positive mutations can be obtained by multiple rounds of in situ error-prone PCR. This method has been used to create random mutagenesis libraries of a xylanase gene and a cellulase gene. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PCR for creating random mutagenesis libraries for directed evolution.