Extrachromosomal DNA (abbreviated ecDNA) is any DNA that is found off the chromosomes, either inside or outside the nucleus of a cell. Most DNA in an individual genome is found in chromosomes contained in the nucleus. Multiple forms of extrachromosomal DNA exist, and, while some of these serve important biological functions, they can also play a role in diseases such as cancer.

In prokaryotes, nonviral extrachromosomal DNA is primarily found in plasmids, whereas, in eukaryotes extrachromosomal DNA is primarily found in organelles. Mitochondrial DNA is a main source of this extrachromosomal DNA in eukaryotes. The fact that this organelle contains its own DNA supports the hypothesis that mitochondria originated as bacterial cells engulfed by ancestral eukaryotic cells. Extrachromosomal DNA is often used in research into replication because it is easy to identify and isolate.

Although extrachromosomal circular DNA (eccDNA) is found in normal eukaryotic cells, extrachromosomal DNA (ecDNA) is a distinct entity that has been identified in the nuclei of cancer cells and has been shown to carry many copies of driver oncogenes. ecDNA is considered to be a primary mechanism of gene amplification, resulting in many copies of driver oncogenes and very aggressive cancers.  

Extrachromosomal DNA in the cytoplasm has been found to be structurally different from nuclear DNA. Cytoplasmic DNA is less methylated than DNA found within the nucleus. It was also confirmed that the sequences of cytoplasmic DNA were different from nuclear DNA in the same organism, showing that cytoplasmic DNAs are not simply fragments of nuclear DNA. In cancer cells, ecDNA have been shown to be primarily isolated to the nucleus (reviewed in ).

In addition to DNA found outside the nucleus in cells, infection by viral genomes also provides an example of extrachromosomal DNA.