Evolutionary innovation relies partially on changes in gene regulation. While a growing body of evidence demonstrates that such innovation is generated by functional changes or translocation of regulatory elements via mobile genetic elements, the de novo generation of enhancers from non-regulatory/non-mobile sequences has, to our knowledge, not previously been demonstrated. Here we show evidence for the de novo genesis of enhancers in vertebrates. For this, we took advantage of the massive gene loss following the last whole genome duplication in teleosts to systematically identify regions that have lost their coding capacity but retain sequence conservation with mammals. We found that these regions show enhancer activity while the orthologous coding regions have no regulatory activity. These results demonstrate that these enhancers have been de novo generated in fish. By revealing that minor changes in non-regulatory sequences are sufficient to generate new enhancers, our study highlights an important playground for creating new regulatory variability and evolutionary innovation.