We have developed a procedure that enables visualization of the genomic loci that are bound by complexes formed by a specific combination of chromatin-binding proteins. This procedure is based on imaging bimolecular fluorescence complementation (BiFC) complexes on Drosophila polytene chromosomes. BiFC complexes are formed by the facilitated association of two fluorescent protein fragments that are fused to proteins that interact with, or are in close proximity to, each other. The intensity of BiFC complex fluorescence at individual genomic loci is greatly enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. The loci that are bound by the complexes are mapped by comparing the locations of BiFC complex fluorescence with the stereotypical banding patterns of the chromosomes. We describe strategies for the design, expression, and validation of fusion proteins for the analysis of BiFC complex binding on polytene chromosomes. We detail protocols for the preparation of polytene chromosome spreads that have been optimized for the purpose of BiFC complex visualization. Finally, we provide guidance for the interpretation of results from studies of BiFC complex binding on polytene chromosomes and for comparison of the genomic loci that are bound by BiFC complexes with those that are bound by subunits of the corresponding endogenous complexes. The visualization of BiFC complex binding on polytene chromosomes provides a unique method to visualize multiprotein complex binding at specific loci, throughout the genome, in individual cells.