Over the past decade, the focus of cloning has shifted from constructing plasmids that express a single gene of interest to creating multigenic constructs that contain entire pathways or even whole genomes. Traditional cloning methods that rely on restriction digestion and ligation are limited by the number and size of fragments that can efficiently be combined. Here, we focus on the use of homologous-recombination-based DNA manipulation in the yeast Saccharomyces cerevisiae for the construction of plasmids from multiple DNA fragments. Owing to its simplicity and high efficiency, cloning by homologous recombination in yeast is very accessible and can be applied to high-throughput construction procedures. Its applications extend beyond yeast-centered purposes and include the cloning of large mammalian DNA sequences and entire bacterial genomes.