Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample (such as blood or tissue) is digested with restriction enzymes, and the resulting DNA fragments are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

The Southern blotting combines the transfer of electrophoresis-separated DNA fragments to a filter membrane in a process called blotting, and the subsequent fragment detection by probe hybridization.

The method is named after the British biologist Edwin Southern, who first published it in 1975. Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named about Edwin Southern's name. As the label is eponymous, Southern is capitalized, as is conventional of proper nouns. The names for other blotting methods may follow this convention, by analogy.