Johannes Heinrich Matthaei (born 4 May 1929) is a German biochemist.

Whilst a post-doctoral visitor in the laboratory of Marshall Warren Nirenberg at the NIH in Bethesda, Maryland, he discovered that a synthetic RNA polynucleotide, composed of a repeating uridylic acid residue (Uracil), coded for a polypeptide chain encoding just one kind of amino acid, phenylalanine.

It was for this and later work on the genetic code for which Nirenberg shared the Nobel Prize for Medicine and Physiology.

In addition, Matthaei and his co-workers in the following years published a multitude of results concerning the early understanding of the form and function of the genetic code.

Later, Matthaei was a member of the Max Planck Society in Göttingen as a director.