The recent completion of the human genome sequence has enabled the identification of a large fraction of our gene catalogue and their physical chromosomal position. However, current efforts lag at defining the cis-regulatory sequences that control the spatial and temporal patterns of each gene’s expression. This task remains difficult due to our lack of knowledge of the vocabulary controlling gene regulation and the vast genomic search space, with greater than 95% of our genome being noncoding. Recent comparative genomic-based strategies are beginning to aid in the identification of functional sequences based on their high levels of evolutionary conservation. This has proven successful for comparisons between closely related species such as human-primate or human-mouse, but also holds true for distant evolutionary comparisons, such as human-fish or human-bird. In this review we provide support for the utility of cross-species sequence comparisons by illustrating several applications of this strategy, including the identification of new genes and functional non-coding sequences. We also discuss emerging concepts as this field matures, such as how to properly select which species for comparison, which may differ significantly between independent studies.