A complementation test is a simple cross to identify whether the two mutations are located on the same gene or different genes. It was first performed by Edward Lewis in the 1940s while working on fruit flies. He developed the test to identify the location and arrangement of different mutations on chromosomes.

Organisms heterozygous for different mutations are crossed pairwise in all combinations. If present on different genes, the mutations can complement each other by providing the missing genetic element producing a wild-type phenotype. However, if both mutations belong to the same gene, they cannot complement each other and fail to exhibit the wild-type phenotype. The mutations that fail to complement each other are said to be in the same complementation group. The mutations that complement each other belong to different complementation groups.