AbstractRecent studies reveal that de novo gene origination from previously non-genic sequences is a common mechanism for gene innovation. These young genes provide an opportunity to study the structural and functional origins of proteins. Here, we combine high-quality base-level whole-genome alignments and computational structural modeling to study the origination, evolution, and protein structures of lineage-specific de novo genes. We identify 555 de novo gene candidates in D. melanogaster that originated within the Drosophilinae lineage. Sequence composition, evolutionary rates, and expression patterns indicate possible gradual functional or adaptive shifts with their gene ages. Surprisingly, we find little overall protein structural changes in candidates from the Drosophilinae lineage. We identify several candidates with potentially well-folded protein structures. Ancestral sequence reconstruction analysis reveals that most potentially well-folded candidates are often born well-folded. Single-cell RNA-seq analysis in testis shows that although most de novo gene candidates are enriched in spermatocytes, several young candidates are biased towards the early spermatogenesis stage, indicating potentially important but less emphasized roles of early germline cells in the de novo gene origination in testis. This study provides a systematic overview of the origin, evolution, and protein structural changes of Drosophilinae-specific de novo genes.