<p>De novo genes generally refer to genes that arise from previously non-coding sequences. This evolutionary path — when randomly expressed sequences become folded and active proteins — challenges our understanding of genetic innovation and has prompted studies to address the evolutionary and mechanistic knowledge gaps. More specifically, prior work has illuminated the mechanisms underlying the origin of de novo genes, their potential functional roles in the cell and the evolutionary processes that lead to these functions. Recent advances in both experimental and computational approaches have contributed to insights into the emergence of de novo genes and the broader implications for our understanding of biological complexity. In this Review, we place particular emphasis on efforts to quantify the likelihood of de novo gene emergence in eukaryotes given genomic characteristics, as well as the mechanisms by which de novo protein structures that are not actively selected against become amenable to selection-driven changes.</p>