<title>Author Summary</title> <p>Gene duplication may facilitate the acquisition of genetic diversity. Little is known, however, about the impact of gene loss on the functions of surviving genes. When a gene is lost, can other closely related genes evolve to perform the functions of the lost gene? Answering this question can be difficult because the proof for gene loss is based on negative evidence and thus can easily pass unnoticed. Here, we illustrate how the comparison of genomic neighborhoods in different species can help reconstruct the chromosomal history of a gene family and provide robust evidence for gene loss, even without an appropriate early-diverging comparator group. Identifying gene loss is important because it helps distinguish between gene gain as a lineage-specific innovation and gene loss as a lineage-specific simplification. As a case study, we investigated the expression of the <italic>Aldh1a</italic> family, which is crucial for retinoic acid signaling in development of eyes, limbs, the brain, and in cancer. Results showed that gene loss is indeed associated with the evolution of functional change in surviving gene family members. Our results highlight the relevance of comparative genomics for identifying gene loss and improving the functional connectivity among human and model organism genomes.</p>