Regulatory subfunctionalization is considered to be a major mechanism for the retention of gene duplicates in the genome. This work provides the first large-scale identification and analysis of putative cis-regulatory elements through comparative genomics between duplicated genes using the Fugu genome as a model. Using seven pairs of fish-specific gene duplicates we showed that all pairs have undergone a level of element partition consistent with one of the main mechanisms proposed for regulatory subfunctionalization. In addition, the regulatory elements in this study may have undergone more subtle levels of subfunctionalization through differential loss of element content and asymmetrical rates of substitution. In addition to presenting this work as an analysis in its own right, the methods in this study can be extended to any similar study in which regions derived from an intra-genomic duplication can be compared to one or more related genomes in which the orthologous region has remained single-copy.