An overlapping gene (or OLG) is a gene whose expressible nucleotide sequence partially overlaps with the expressible nucleotide sequence of another gene. In this way, a nucleotide sequence may make a contribution to the function of one or more gene products. Overlapping genes are present in and a fundamental feature of both cellular and viral genomes. The current definition of an overlapping gene varies significantly between eukaryotes, prokaryotes, and viruses. In prokaryotes and viruses overlap must be between coding sequences but not mRNA transcripts, and is defined when these coding sequences share a nucleotide on either the same or opposite strands. In eukaryotes, gene overlap is almost always defined as mRNA transcript overlap. Specifically, a gene overlap in eukaryotes is defined when at least one nucleotide is shared between the boundaries of the primary mRNA transcripts of two or more genes, such that a DNA base mutation at any point of the overlapping region would affect the transcripts of all genes involved. This definition includes 5′ and 3′ untranslated regions (UTRs) along with introns.

Overprinting refers to a type of overlap in which all or part of the sequence of one gene is read in an alternate reading frame from another gene at the same locus. The alternative open reading frames (ORF) are thought to be created by critical nucleotide substitutions within an expressible pre-existing gene, which can be induced to express a novel protein while still preserving the function of the original gene. Overprinting has been hypothesized as a mechanism for de novo emergence of new genes from existing sequences, either older genes or previously non-coding regions of the genome. It is believed that most overlapping genes, or genes whose expressible nucleotide sequences partially overlap with each other, evolved in part due to this mechanism, suggesting that each overlap is composed of one ancestral gene and one novel gene. Subsequently, overprinting is also believed to be a source of novel proteins, as de novo proteins coded by these novel genes usually lack remote homologs in databases. Overprinted genes are particularly common features of the genomic organization of viruses, likely to greatly increase the number of potential expressible genes from a small set of viral genetic information. It is likely that overprinting is responsible for the generation of numerous novel proteins by viruses over the course of their evolutionary history.

Genes may overlap in a variety of ways and can be classified by their positions relative to each other.

Overlapping genes can also be classified by phases, which describe their relative reading frames:

Studies on overlapping genes suggest that their evolution can be summarized in two possible models. In one model, the two proteins encoded by their respective overlapping genes evolve under similar selection pressures. The proteins and the overlap region are highly conserved when strong selection against amino acid change is favored. Overlapping genes are reasoned to evolve under strict constraints as a single nucleotide substitution is able to alter the structure and function of the two proteins simultaneously. A study on the hepatitis B virus (HBV), whose DNA genome contains numerous overlapping genes, showed the mean number of synonymous nucleotide substitutions per site in overlapping coding regions was significantly lower than that of non-overlapping regions. The same study showed that it was possible for some of these overlapping regions and their proteins to diverge significantly from the original when there's weak selection against amino acid change. The spacer domain of the polymerase and the pre-S1 region of a surface protein of HBV, for example, had a percentage of conserved amino acids of 30% and 40%, respectively. However, these overlap regions are known to be less important for replication compared to the overlap regions that were highly conserved among different HBV strains, which are absolutely essential for the process.

The second model suggests that the two proteins and their respective overlap genes evolve under opposite selection pressures: one frame experiences positive selection while the other is under purifying selection. In tombusviruses, the proteins p19 and p22 are encoded by overlapping genes that form a 549 nt coding region, and p19 is shown to be under positive selection while p22 is under purifying selection. Additional examples are mentioned in studies involving overlapping genes of the Sendai virus, potato leafroll virus, and human parvovirus B19. This phenomenon of overlapping genes experiencing different selection pressures is suggested to be a consequence of a high rate of nucleotide substitution with different effects on the two frames; the substitutions may be majorly non-synonymous for one frame while mostly being synonymous for the other frame.

Overlapping genes are particularly common in rapidly evolving genomes, such as those of viruses, bacteria, and mitochondria. They may originate in three ways:

The use of the same nucleotide sequence to encode multiple genes may provide evolutionary advantage due to reduction in genome size and due to the opportunity for transcriptional and translational co-regulation of the overlapping genes. Gene overlaps introduce novel evolutionary constraints on the sequences of the overlap regions.