LAGLIDADG homing endonucleases (LHEs) are valuable tools for genome engineering, and our ability to alter LHE target site specificity is rapidly evolving. However, widespread use of these enzymes is limited due to the small number of available engineering scaffolds, each requiring extensive redesign to target widely varying DNA sequences. Here, we describe a technique for the chimerization of homologous I-OnuI family LHEs. Chimerization greatly expands the pool of unique starting scaffolds, thereby enabling more effective and efficient LHE redesign. I-OnuI family enzymes are divided into N- and C-terminal halves based on sequence alignments, and then combinatorially rejoined with a hybrid linker. The resulting chimeric enzymes are expressed on the surface of yeast where stability, DNA binding affinity, and cleavage activity can be assayed by flow cytometry.