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Designer nucleases have revolutionized the study of common model organisms by enabling the simple and efficient manipulation of specified genomic sequences. However, therapeutic application of this technology may be limited by predictable and unanticipated off-target lesions that may be prevalent with all nuclease platforms to lesser and greater extents. This limitation may become even more problematic when desired targets are homologous to other genome sequences. Here we report the development of a multi-reporter selection system for the generation of nucleases that can discriminate between highly homologous targets. We have applied this system to create zinc finger nuclease (ZFNs) that can discriminate between highly homologous sequences from the CCR5 and CCR2 genes. These nucleases were tested in human cell culture and demonstrate strong on-target activity (≈10-60% indel frequency) with minimal or no nuclease activity at CCR2. Further, an unbiased screen of off-target activity demonstrates excellent genome-wide fidelity. In one case, the nuclease pair results in only one off-target with greater than 0.1% indel frequency (0.37%) and an aggregate off-target activity of 0.5%. This system offers a simple approach to generate nucleases, zinc finger or otherwise, with exceptional genome-wide fidelity regardless of target.