Assembly of combinatorial genetic libraries for identification of biomolecules with novel or improved properties requires high fidelity and efficiency to produce the greatest spectrum of genetically diverse clones. We have been developing homologous recombination (HR) as a platform for the production of combinatorial genetic libraries for affinity maturation and diversification of human therapeutic antibodies. Therapeutic antibodies have a great clinical potential in various therapeutic settings including the treatment of a number of oncology, autoimmune and infectious diseases, organ transplantation, and others. In brief, we describe a method, where CDR-encoding DNA oligos and a gapped vector containing the heavy and light chain genes are cotransformed into budding yeast Saccharomyces cerevisiae for in vivo assembly by HR. Importantly, the affinity of resulting antibody clones in the generated library can be directly assayed by yeast surface display without subcloning and retransformation. Furthermore, mating two haploid yeast strain libraries each encoding a variation of heavy chain or light chain genes enables fast screening of the heavy/light chain combinations displayed by the resulting diploids. Finally, this method can be generalized to generate combinatorial genetic libraries for other applications.