The introduction of defined mutations into open reading frames (ORF) or non‐translated regions of the genome is important to study of the structure–function relationship of amino acid residues in proteins or that of sequence motifs at the genome level. We describe a simple two‐step method for the introduction of defined single or multiple point mutations into the genome of Saccharomyces cerevisiae. This method circumvents the need for plasmid‐based mutagenesis and thus ensures homogenous expression of the gene of interest within the cell population. It is based on the introduction of a selectable marker downstream of the gene of interest. This marker is then amplified with a gene‐specific primer that harbours the desired point mutation, creating a selectable marker‐tagged mutant version of the gene of interest. The mutant fragment is then integrated into the genome of a wild‐type strain through homologous recombination. Successive rounds of amplification of the mutant loci with primers that introduce additional point mutations upstream of existing mutations will generate multiple defined mutations within a single gene. As a proof of principle, we have employed this method to generate a temperature‐sensitive mutant version of the plasma membrane ATPase, pma1‐7, which bears two point mutations (Pro434Ala and Gly789Ser). Phenotypic analysis of a pma1‐7 haploid strain indicates that this allele has the same characteristics as the original pma1‐7 allele. It confers a temperature‐sensitive growth phenotype and the newly synthesized Pma1‐7 protein is unstable and rapidly degraded. Copyright © 2006 John Wiley & Sons, Ltd.